Phylum XVI. Tenericutes Murray 1984a, 356VP (Effective publication: Murray 1984b, 33.) Da n i e l R. Br o w n Ten.er¢i.cutes. L. adj. tener tender; L. fem. n. cutis skin; N.L. fem. n. Tenericutes prokaryotes of a soft pliable nature indicative of a lack of a rigid cell wall.

Members of the Tenericutes are wall-less bacteria that do not syn- the ­organisms are evolutionarily related to certain clostridia, thesize precursors of peptidoglycan. the absence of a cell wall cannot be equated with Gram reac- tion positivity or with other members of the Firmicutes. It is Further descriptive information unfortunate that workers involved in determinative bacteriology The nomenclatural type by monotypy (Murray, 1984a) is the have a reference in which wall-free prokaryotes are described as class Mollicutes, which consists of very small prokaryotes that Gram-positive bacteria” (Tully, 1993a). Despite numerous valid are devoid of cell walls. Electron microscopic evidence for the assignments of novel species of mollicutes to the Tenericutes dur- absence of a cell wall was mandatory for describing novel species ing the intervening years, the class Mollicutes was still included of mollicutes until very recently. Genes encoding the pathways in the phylum Firmicutes in the most recent revision of the Taxo- for peptidoglycan biosynthesis are absent from the genomes of nomic Outline of Bacteria and Archaea (TOBA), which is based more than 15 species that have been annotated to date. Some solely on the phylogeny of 16S rRNA genes (Garrity et al., 2007). species do possess an extracellular glycocalyx. The absence of The taxon Tenericutes is not recognized in the TOBA, although a cell wall confers such mechanical plasticity that most molli- paradoxically it is the phylum consisting of the Mollicutes in the cutes are readily filterable through 450 nm pores and many spe- most current release of the Ribosomal Database Project (Cole cies have some cells in their populations that are able to pass et al., 2009). Mollicutes are specifically excluded from the most through 220 nm or even 100 nm filters. However, they may vary recently emended description of the Firmicutes in Bergey’s Manual in shape from coccoid to flask-shaped cells or helical filaments of ­Systematic Bacteriology (2nd edition, volume 3; De Vos et al., that reflect flexible cytoskeletal elements. 2009) on the grounds of their lack of rigid cell walls plus analyses of strongly supported alternative universal phylogenetic markers, Taxonomic comments including RNA polymerase subunit B, the chaperonin GroEL, To provide greater definition and formal nomenclature for several different aminoacyl tRNA synthetases, and subunits of vernacular names used in the 8th edition of Bergey’s Manual of F0F1-ATPase (Ludwig et al., 2009; Ludwig and Schleifer, 2005). Determinative Bacteriology (Bergey VIII; Buchanan and Gibbons, The taxonomic dignity of Tenericutes bestowed by its original 1974), Gibbons and Murray (1978) proposed that the higher formal validation, and upheld by a quarter of a century of valid taxa of prokaryotes be subdivided primarily according to the descriptions of novel species of mollicutes, has therefore been presence and character, or absence, of a rigid or semirigid cell respected in this volume of Bergey’s Manual. wall as reflected in the determinative Gram reaction. Similar Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, to the non-hierarchical groupings of Bergey VIII, which were Bové, Laigret and Whitcomb 1993b, 382. based on a few readily determined criteria, the “wall-deficient” organisms grouped together in the first edition of The Prokary- References otes included the mollicutes (Starr et al., 1981). While acknowl- Buchanan, R.E. and N.E. Gibbons (editors). 1974. Bergey’s Manual of edging the emerging 16S rRNA-based evidence that indicated Determinative Bacteriology, 8th edn. Williams & Wilkins, Baltimore. a phylogenetic relationship between mollicutes and certain Cole, J.R., Q. Wang, E. Cardenas, J. Fish, B. Chai, R.J. Farris, A.S. Kulam- Gram-stain-positive bacteria in the division Firmicutes, Murray Syed-Mohideen, D.M. McGarrell, T. Marsh, G.M. Garrity and J.M. (1984b) proposed the separate division Tenericutes for the stable Tiedje. 2009. The Ribosomal Database Project: improved alignments and distinctive group of wall-less species that are not simply an and new tools for rRNA analysis. Nucleic Acids Res. 37: (Database obvious subset of the Firmicutes. issue): D141–D145. The approved divisional rank of Tenericutes and the assignment De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. of class Mollicutes as its nomenclatural type (Murray, 1984a) were Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Systematic adopted by the International Committee on Systematic Bacte- Bacteriology, 2nd edn, vol. 3. Springer, New York. Freundt, E.A. 1955. The classification of the pleuropneumoniae group riology’s Subcommittee on the Taxonomy of Mollicutes (Tully, of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. 1988) and subsequent valid taxonomic descriptions assigned 5: 67–78. novel species of mollicutes to the Tenericutes. However, the second Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and B.J. (1992) and third (2007) editions of The Prokaryotes described the Tindall. 2007. The Taxonomic Outline of the Bacteria and Archaea, mollicutes instead as Firmicutes with low G+C DNA. The Subcom- Release 7.7, Part 11 – The Bacteria: Phyla Planctomycetes, Chlamyd- mittee considered this to be an unfortunate ­grouping: “While iae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, Fusobacteria,

567 568 Phylum XVI. Tenericutes

­Verrucomicrobia, Dictyoglomi, Gemmatomonadetes, and Lentisphaerae. pp. Starr, M.P., H. Stolp, H.G. Trüper, A. Balows and H.G. Schlegel ­(editors). 540–595. (http://www.taxonomicoutline.org/). 1981. The Prokaryotes. Springer, Berlin. Gibbons, N.E. and R.G.E. Murray. 1978. Proposals concerning the Tully, J.G. 1988. International Committee on Systematic Bacteriol- higher taxa of bacteria. Int. J. Syst. Bacteriol. 28: 1–6. ogy, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacte- Interim Meeting, 25 and 28 August 1986, Birmingham, Alabama. Int. ria based on comparative sequence analysis of conserved genes. In J. Syst. Bacteriol. 38: 226–230. Microbial Phylogeny and Evolution, Concepts and Controversies, Tully, J.G. 1993a. International Committee on Systematic Bacteriol- (edited by Sapp). Oxford University Press, New York, pp. 70–98. ogy, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Ludwig, W., K.H. Schleifer and W.B. Whitman. 2009. Revised road map to Interim Meetings, 1 and 2 August, 1992, Ames, Iowa. Int. J. Syst. the phylum Firmicutes. In Bergey’s Manual of Systematic Bacteriology, 2nd ­Bacteriol. 43: 394–397. edn, vol. 3, The Firmicutes (edited by De Vos, Garrity, Jones, Krieg, Lud- Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993b. Revised wig, Rainey, Schleifer and Whitman). Springer, New York, pp. 1–13. ­taxonomy of the class Mollicutes – proposed elevation of a monophyl- Murray, R.G.E. 1984a. In Validation of the publication of new names etic cluster of arthropod-associated mollicutes to ordinal rank (Ento- and new combinations previously effectively published outside the moplasmatales ord. nov.), with provision for familial rank to separate IJSB. List no. 15. Int. J. Syst. Bacteriol. 34: 355–357. species with nonhelical morphology (Entomoplasmataceae fam. nov.) Murray, R.G.E. 1984b. The higher taxa, or, a place for everything…? In from helical species (Spiroplasmataceae), and emended descriptions Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. and Holt). Williams & Wilkins, Baltimore, pp. 31–34. ­Bacteriol. 43: 378–385.

Class I. Mollicutes Edward and Freundt 1967, 267AL

Da n i e l R. Br o w n , Me g h a n Ma y , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n Mol¢li.cutes or Mol.li.cu¢tes. L. adj. mollis soft, pliable; L. fem. n. cutis skin; N.L. fem. pl. n. Mollicutes class with pliable cell boundary.

Very small prokaryotes totally devoid of cell walls. Bounded pathogens of humans, animals, insects, or plants. Genome sizes by a plasma membrane only. Incapable of synthesis of pepti- range from 580 to 2200 kbp, among the smallest recorded in doglycan or its precursors. Consequently resistant to penicillin prokaryotes. The genomes of more than 20 species have been and its derivatives and sensitive to lysis by osmotic shock, deter- completely sequenced and annotated to date (Table 135). The gents, alcohols, and specific antibody plus complement. Gram- G+C content of the DNA is usually low, ~23–34 mol%, but in some stain-negative due to lack of cell wall, but constitute a distinct species is as high as ~40 mol% (Bd, Tm). Can be distinguished from phylogenetic lineage within the Gram-stain-positive bacteria other bacteria in having only one or two rRNA operons (one spe- (Woese et al., 1980). Pleomorphic, varying from spherical or cies of Mesoplasma has three) and an RNA polymerase that is resis- flask-shaped structures to branched or helical filaments. The tant to rifampin. The 5S rRNA contains fewer nucleotides than coccoid and flask-shaped cells usually range from 200–500 nm that of other bacteria and there are fewer tRNA genes. In some in diameter, although cells as large as 2000 nm have been seen. genera, instead of a stop, the UGA codon encodes tryptophan. Replicate by binary fission, but genome replication may pre- Plasmids and viruses (phage) occur in some species. cede cytoplasmic division, leading to the formation of multi- Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, nucleated filaments. Colonies on solid media are very small, Bové, Laigret and Whitcomb 1993, 382. usually much less than 1 mm in diameter. The organisms tend to penetrate and grow inside the solid medium. Under suitable Further descriptive information conditions, almost all species form colonies that have a char- Table 136 summarizes the present classification of the Mollicutes acteristic fried-egg appearance. Usually nonmotile, but some into families and genera and provides the major distinguishing species show gliding motility. Species that occur as helical fila- characteristics of these taxa. The trivial term mycoplasma has ments show rotary, flexional, and translational motility. No rest- been used to denote any species included in the class Mollicutes, ing stages are known. but the term mollicute(s) is now considered most appropriate The species recognized so far can be grown on artificial cell- as the trivial name for all members of the class, so that the triv- free media of varying complexity, although certain strains may ial name mycoplasma can be retained only for members of the be more readily isolated by cell-culture procedures. Many “Can- genus Mycoplasma. Hemotropic mycoplasmas are referred to by didatus” species have been proposed and characterized at the the trivial name hemoplasmas. The trivial names ureaplasma, molecular level, but not yet cultivated axenically. Most cultiva- entomoplasma, mesoplasma, spiroplasma, acholeplasma, ble species require sterols and fatty acids for growth. However, anaeroplasma, and asteroleplasma are commonly used when members of some genera can grow well in either serum-free reference is made to members of the corresponding genus. media or serum-free media supplemented with polyoxyethyl- Their 16S rRNA gene sequences usefully place the mol- ene sorbitan. Most species are facultative anaerobes, but some licutes into phylogenetic groups ( Johansson, 2002; Weisburg are obligate anaerobes that are killed by exposure to minute et al., 1989) and an analysis of 16S rRNA gene sequences is quantities of oxygen. No tricarboxylic acid cycle enzymes, qui- now mandatory for characterization of novel species (Brown nones, or cytochromes have been found. et al., 2007). 16S rRNA gene sequences have also shown that All mollicutes are commensals or parasites, occurring in a wide certain ­hemotropic bacteria, previously considered to be range of vertebrate, insect, and plant hosts. Many are significant­ ­members of the Rickettsia, belong to the order Mycoplasmatales. Table 135. Characteristics of sequenced mollicute genomesa d b c c Ureaplasma urealyticum Ureaplasma Acholeplasma laidlawii “Candidatus Phytoplasma asteris” Species Mycoplasma agalactiae Mycoplasma arthritidis Mycoplasma capricolum subsp. capricolum Mycoplasma conjunctivae Mycoplasma gallisepticum Mycoplasma genitalium Mycoplasma hominis Mycoplasma hyopneumoniae Mycoplasma mobile Mycoplasma mycoides subsp. mycoides SC Mycoplasma penetrans Mycoplasma pneumoniae Mycoplasma pulmonis Mycoplasma synoviae Mesoplasma florum parvumUreaplasma

Strain PG2T 158L3-1 ATCC HRC/581T R G-37T PG21T JT 163KT PG1T HF-2 M129 UAB CTIP 53 L1T ATCC ATCC PG8T AYWB 27343T 700970 33699 Size (mb) 0.88 0.82 1.0 0.85 0.99 0.58 0.67 0.90 0.78 1.21 1.36 0.82 0.96 0.80 0.79 0.75 0.87 1.5 0.71 DNA G+C 29 30 23 28 31 31 27 28 25 24 26 40 26 28 27 25 25 31 26 content (mol%) Open reading 742 631 812 727 726 475 537 657 633 1016 1037 689 782 659 687 653 692 1433 671 frames Hypothetical 50 nd nd 45 37 21 36 nd 27 41 41 38 38 33 nd 33 nd nd nd genes (%) Coding 87 80 88 90 91 90 90 87 90 80 88 87 90 91 92 91 89 90 73 density (%) References Sirand- Dybvig J. Glass Calderon- Papazisi Fraser Pereyre Vasconcelos Jaffe Westberg Sasaki Himmelreich Cham­baud Vasconcelos Knight Glass na na Bai Pugnet et al. and Copete et al. et al. et al. et al. et al. et al. et al. et al. et al. (2001) et al. et al. et al. et al. et al. (2008) others, et al. (2003) (1995) (2009) (2005) (2004) (2004) (2002) (1996) (2005) (2004) (2000) (2006) (2007) unpub- (2009) lished. and, Not determined; na, not available. bMycoplasma hyopneumoniae strains 232 and 7448 were sequenced by Minion et al. (2004) and Vasconcelos et al. (2005). cData for Ureaplasma parvum and Ureaplasma urealyticum refer to serovars 3 and 10, respectively; serovars 1–14 were sequenced and deposited directly into GenBank. dAYWB, Aster yellows witches’ broom; the onion yellows strain was sequenced by Oshima et al. (2004). 570 Phylum XVI. Tenericutes Hemotropic Hemotropic Urea hydrolysis Growth with PES Defining features Strictly anaerobic Strictly anaerobic Helical morphology Not yet cultured in vitro c A A A A N, P N, P N, P N, P H, A H, A A, N, P Habitat − + − + − + + + nd nd nd Cholesterol requirement Nd Nd 1,500 825–930 870–900 530–1,350 780–2,220 760–1,140 580–1,350 1,500–1,600 1,500–1,650 Genome size range (kbp) b Species 1, 0, 0 4, 0, 0 7, 0, 0 4, 0, 0 1, 0, 0 6, 0, 0 0, 27, 0 11, 0, 0 37, 0, 0 18, 0, 0 116, 9, 1, 4 Genus Asteroleplasma Anaeroplasma “ Candidatus Phytoplasma” Mesoplasma Spiroplasma Acholeplasma Ureaplasma Eperythrozoon Haemobartonella Entomoplasma Mycoplasma a Family Anaeroplasmataceae Anaeroplasmataceae Incertae sedis Entomoplasmataceae Spiroplasmataceae Acholeplasmataceae Mycoplasmataceae Incertae sedis Incertae sedis Entomoplasmataceae Mycoplasmataceae d d Description of the class Mollicutes

Affiliation of the constituent genera within Mycoplasmatales has not been formalized. Numbers of species: valid, Candidatus , incertae sedis , invalid. nd, Not determined; PES, polyoxyethylene sorbitan. H, human; A, vertebrate animal; N, invertebrate animal; P, plant. H, human; A, vertebrate animal; N, invertebrate P, IV. Anaeroplasmatales IV. IV. Anaeroplasmatales IV. III. Acholeplasmatales II. Entomoplasmatales II. Entomoplasmatales III. Acholeplasmatales I. Mycoplasmatales I. Mycoplasmatales I. Mycoplasmatales II. Entomoplasmatales e 136. Tabl Order a b c d I. Mycoplasmatales Phylum XVI. Tenericutes 571

The phytoplasmas, a large group of uncultivated mollicutes a branch of the Firmicutes. The initial event in this evolution occurring as agents that can cycle between plant and inver- was proposed to be the formation of the Acholeplasma branch, tebrate hosts, have been given a provisional “Candidatus Phy- although the position of the Anaeroplasma species (Anaero- toplasma” genus designation. The 16S rRNA gene sequences plasma bactoclasticum and Anaeroplasma abactoclasticum) was not from at least ten unique phylotypes, recently discovered among definitely established within these dendrograms. Formation the human microbial flora through global 16S rRNA gene PCR of the acholeplasmas may have coincided with a reduction in (Eckburg et al., 2005), cluster distinctly enough to suggest the genome size to about 1500–1700 kb and loss of the cell wall. existence of a yet-uncircumscribed order within the class (May With a genome size similar to the acholeplasmas, the spiroplas- et al., 2009). mas may have formed from the acholeplasmas. Later indepen- Non-helical mollicutes isolated from insects and plants have dent genome reductions to 500–1000 kb may have led to the been placed in the order Entomoplasmatales, the two genera origins of the sterol-requiring mycoplasma and ureaplasma lin- of which are distinguished by their requirement for choles- eages. The more extensive phylogenetic analysis of Weisburg terol (Tully et al., 1993). Members of the genus Entomoplasma et al. (1989) examined the 16S rRNA gene sequences of about require cholesterol; those of the genus Mesoplasma do not. 50 species of mollicutes and confirmed a number of these However, sterol requirements do not correlate well with phy- observations and provided additional insights into mollicute logenetic analyses in other groups. At least four species of evolution. These results also indicated that the acholeplasmas spiroplasmas do not require sterol for growth, but they do not formed upon the initial divergence of mollicutes from clostrid- form a phylogenetic group. Within the order including the ial ancestors. Further divergence of this stem led to the sterol- obligately anaerobic mollicutes Anaeroplasmatales, members requiring, anaerobic Anaeroplasma and the non-sterol-requiring of the genus Anaeroplasma require sterols for growth, whereas Asteroleplasma branches. The Spiroplasma branch also appeared members of the genus Asteroleplasma do not (Robinson et al., to originate from within the acholeplasmas, with further evolu- 1975; Robinson and Freundt, 1987). Thus, sterol requirement tion leading to a series of repeated and independent genome is a useful phylogenetic marker only in the Acholeplasmatales reductions from nearly 2000 kb to 600–1200 kb to yield the and Anaeroplasmatales. Mycoplasma and Ureaplasma lineages. In the past, there was some risk of confusing mollicutes with Based on the phylogeny of 16S rRNA genes, the class wall-less “L (Lister)-phase” variants of certain other bacteria, but Mollicutes­ was included in the phylum Firmicutes in the most simple PCR-based analyses of 16S rRNA or other gene sequences recent revision of the Taxonomic Outline of Bacteria and Archaea now obviate that concern. Wall-less members of the genus Ther- ­(Garrity et al., 2007). However, the Mollicutes are excluded moplasma, previously assigned to the Mollicutes, are Archaea and from the most recently emended description of the Firmicutes differ from all other members of this class in their 16S rRNA (De Vos et al., 2009) based on alternative phylogenetic markers, nucleotide sequences plus a number of important features relat- including RNA polymerase subunit B, the chaperonin GroEL, ing to their mode of life and metabolism. Thus, they are quite several different aminoacyl tRNA synthetases, and subunits of unrelated to this class (Fox et al., 1980; Razin and Freundt, 1984; F0F1-ATPase (Ludwig and Schleifer, 2005). Woese et al., 1980). Members of the Erysipelothrix line of descent, The Weisburg et al. (1989) study also proposed five addi- also formerly assigned to the Mollicutes, are now assigned to the tional phylogenetic groupings within the mollicutes, including class Erysipelotrichi in the phylum Firmicutes (Stackebrandt, 2009; the anaeroplasma, asteroleplasma, spiroplasma, pneumoniae, Verbarg et al., 2004). and hominis groups (Figure 105). Phytoplasmas are similar to acholeplasmas in their 16S rRNA gene sequences and UGA Taxonomic comments codon usage (IRPCM Phytoplasma/Spiroplasma Working The origin of mollicutes and their relationships to other Team – Phytoplasma Taxonomy Group, 2004). They probably prokaryotes was controversial for many years, especially since diverged from acholeplasmas at about the same time as the split their small genomes and comparative phenotypic simplicity of spiroplasmas into helical and non-helical lineages (Maniloff, suggested that they might have descended from a primitive 2002). The modern species concept for mollicutes is justified organism. The first comparative phylogenetic analysis of the principally by DNA–DNA hybridization, serology, and 16S rRNA origin of mollicutes was carried out by oligonucleotide map- gene sequence similarity (Brown et al., 2007). A large number ping of 16S rRNA gene sequences by Woese et al. (1980). The of individual species have been assigned to phylogenetic groups, organisms then assigned to the genera Mycoplasma, Spiroplasma, clusters, and subclusters that also share other characteristics, and Acholeplasma seemed to have arisen by reductive evolution although the cluster boundaries are sometimes subjective as a deep branch of the clostridial lineage leading to the genera (Harasawa and Cassell, 1996; Johansson, 2002; Pettersson et al., Bacillus and Lactobacillus. This relationship had been proposed 2000, 2001). earlier (Neimark, 1979) because the low G+C mollicutes, strep- Lastly, the type order Mycoplasmatales is assigned to the class tococci, and lactic acid bacteria share characteristic enzymes. as this clearly appeared to be the intention of Edward and Fre- In particular, acholeplasma and streptococcus aldolases show undt (1967) in their paper entitled “Proposal for Mollicutes as high amino acid sequence similarity. name of the class established for the order Mycoplasmatales”. These findings were generally confirmed by studies of 5S rRNA gene sequences (Rogers et al., 1985), which included a Acknowledgements number of acholeplasmas, anaeroplasmas, mycoplasmas, ure- The lifetime achievements in mycoplasmology and major con- aplasmas, and Clostridium innocuum. Dendrograms constructed tributions to the foundation of this material by Joseph G. Tully from evolutionary distance matrices indicated that the mol- are gratefully acknowledged. Daniel R. Brown and Meghan May licutes form a coherent phylogenetic group that developed as were supported by NIH grant 5R01GM076584. 572 Phylum XVI. Tenericutes

Mycoplasma hominis Ureaplasma urealyticum * Mycoplasma pneumoniae Mycoplasma coccoides Spiroplasma apis Mycoplasma mycoides subsp. mycoides Entomoplasma ellychniae Mesoplasma florum Spiroplasma citri Spiroplasma ixodetis Acholeplasma laidlawii ‘Candidatus Phytoplasma’ strain OY-M Anaeroplasma abactoclasticum Asteroleplasma anaerobium Clostridium innocuum

Scale: 0.1 substitutions/site

Figure 105. Phylogenetic grouping of the class Mollicutes. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type genera, plus representatives of other major clusters within the Mycoplas- matales and Entomoplasmatales and a phytoplasma. Clostridium innocuum was the outgroup. All bootstrap values (100 replicates) are >50% except where indicated (asterisk).

Further reading Taylor-Robinson, D. and J. Bradbury. 1998. Mycoplasma diseases. In Topley and Wilson’s Principles and Practice of Microbiol- Barile, M.F., S. Razin, J.G. Tully and R.F. Whitcomb (Editors). ogy, vol. 3 (edited by Hausler and Sussman). Edward Arnold, 1979, 1985, 1989. The Mycoplasmas (five volumes). Academic London, pp. 1013–1037. Press, New York. Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ure- Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (edi- aplasmas, spiroplasmas, and related organisms. In Topley and tors). 1992. Mycoplasmas: Molecular Biology and Pathogen- Wilson, Principles and Practice of Microbiology, 9th edn, vol. esis. American Society for Microbiology, Washington, D.C. 2 (edited by Balows and Duerden). Arnold Publishers, Lon- Murray, R.G.E. 1984. The higher taxa, or, a place for every- don, pp. 799–827. thing…?. In Bergey’s Manual of Systematic Bacteriology, vol. Tully, J.G. and S. Razin (editors). 1996. Molecular and ­Diagnostic 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, ­Procedures in Mycoplasmology, vol. 2. Academic Press, pp. 31–34. San Diego, CA. Razin, S. and J.G.E. Tully. 1995. Molecular and Diagnostic Proce- dures in Mycoplasmology, vol. 1. Academic Press, San Diego.

References De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K. H. Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Bai, X., J. Zhang, A. Ewing, S.A. Miller, A. Jancso Radek, D.V. Systematic Bacteriology, 2nd edn, vol. 3. Springer, New York. Shevchenko, K. Tsukerman, T. Walunas, A. Lapidus, J.W. Campbell Dybvig, K., C. Zuhua, P. Lao, D.S. Jordan, C.T. French, A.H. Tu and and S.A. Hogenhout. 2006. Living with genome instability: the adap- A.E. Loraine. 2008. Genome of Mycoplasma arthritidis. Infect. Immun. tation of phytoplasmas to diverse environments of their insect and 76: 4000–4008. plant hosts. J. Bacteriol. 188: 3682–3696. Eckburg, P., E. Bik, C. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal stan- S. Gill, K. Nelson and D. Relman. 2005. Diversity of the human intes- dards for description of new species of the class Mollicutes (division tinal microbial flora. Science 308: 1635–1638. Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Edward, D.G.ff. and E.A. Freundt. 1967. Proposal for Mollicutes as name Calderon-Copete, S.P., G. Wigger, C. Wunderlin, T. Schmidheini, J. of the class established for the order Mycoplasmatales. Int. J. Syst. Frey, M.A. Quail and L. Falquet. 2009. The Mycoplasma conjunctivae ­Bacteriol. 17: 267–268. genome sequencing, annotation and analysis. BMC Bioinformatics Fox, G.E., E. Stackebrandt, R.B. Hespell, J. Gibson, J. Maniloff, 10 Suppl 6: S7. T.A. Dyer, R.S. Wolfe, W.E. Balch, R.S. Tanner, L.J. Magrum, Chambaud, I., R. Heilig, S. Ferris, V. Barbe, D. Samson, F. Galis- L.B. Zablen, R. Blakemore, R. Gupta, L. Bonen, B.J. Lewis, D.A. Stahl, son, I. Moszer, K. Dybvig, H. Wroblewski, A. Viari, E.P. Rocha and K.R. Luehrsen, K.N. Chen and C.R. Woese. 1980. The phylogeny of A. Blanchard. 2001. The complete genome sequence of the murine prokaryotes. Science 209: 457–463. respiratory pathogen Mycoplasma pulmonis. Nucleic Acids Res. 29: Fraser, C.M., J.D. Gocayne, O. White, M.D. Adams, R.A. Clayton, R.D. 2145–2153. Fleischmann, C.J. Bult, A.R. Kerlavage, G. Sutton, J.M. Kelley and Phylum XVI. Tenericutes 573

et al. 1995. The minimal gene complement of Mycoplasma genita- Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2000. Updated lium. Science 270: 397–403. phylogenetic description of the Mycoplasma hominis cluster (Weisburg Freundt, E.A. 1955. The classification of the pleuropneumoniae group et al. 1989) based on 16S rDNA sequences. Int. J. Syst. Evol. Micro- of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. biol. 50: 291–301. 5: 67–78. Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2001. Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and Re-evaluation of the classical Mycoplasma lipophilum cluster ­(Weisburg B.J. Tindall. 2007. The Taxonomic Outline of the Bacteria and et al. 1989) and description of two new clusters in the hominis group Archaea, Release 7.7, Part 11 – The Bacteria: phyla Planctomycetes, based on 16S rDNA sequences. Int. J. Syst. Evol. Microbiol. 51: Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, 633–643. ­Fusobacteria, ­Verrucomicrobia, Dictyoglomi, Gemmatomonadetes, and Razin, S. and E.A. Freundt. 1984. The Mollicutes, Mycoplasmatales, and Lentisphaerae­ . pp. 540–595. (http://www.taxonomicoutline.org/). Mycoplasmataceae. In Bergey’s Manual of Systematic Bacteriology, Glass, J.I., E.J. Lefkowitz, J.S. Glass, C.R. Heiner, E.Y. Chen and vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, G.H. Cassell. 2000. The complete sequence of the mucosal pathogen pp. 740–742. Ureaplasma urealyticum. Nature 407: 757–762. Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abac- Harasawa, R. and G.H. Cassell. 1996. Phylogenetic analysis of genes toclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from coding for 16S rRNA in mammalian ureaplasmas. Int. J. Syst. Bacte- rumen. Int. J. Syst. Bacteriol. 25: 173–181. riol. 46: 827–829. Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended clas- Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B.C. Li and R. Herrmann. sification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. 1996. Complete sequence analysis of the genome of the bacterium Rogers, M.J., J. Simmons, R.T. Walker, W.G. Weisburg, C.R. Woese, Mycoplasma pneumoniae. Nucleic Acids Res. 24: 4420–4449. R.S. Tanner, I.M. Robinson, D.A. Stahl, G. Olsen, R.H. Leach and IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma Taxon- J. Maniloff. 1985. Construction of the mycoplasma evolutionary omy Group. 2004. Description of the genus ‘Candidatus Phytoplasma’, tree from 5S rRNA sequence data. Proc. Natl. Acad. Sci. U.S.A. 82: a taxon for the wall-less non-helical prokaryotes that colonize plant 1160–1164. phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255. Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, Jaffe, J.D., N. Stange-Thomann, C. Smith, D. DeCaprio, S. Fisher, J. C. Yoshino, A. Horino, T. Shiba, T. Sasaki and M. Hattori. 2002. Butler, S. Calvo, T. Elkins, M.G. Fitzgerald, N. Hafez, C.D. Kodira, J. The complete genomic sequence of Mycoplasma penetrans, an intra- Major, S. Wang, J. Wilkinson, R. Nicol, C. Nusbaum, B. Birren, H.C. cellular bacterial pathogen in humans. Nucleic Acids Res. 30: Berg and G.M. Church. 2004. The complete genome and proteome 5293–5300. of Mycoplasma mobile. Genome Res. 14: 1447–1461. Sirand-Pugnet, P., C. Lartigue, M. Marenda, D. Jacob, A. Barre, V. Barbe, Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular Biology C. Schenowitz, S. Mangenot, A. Couloux, B. Segurens, A. de Daru- and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). var, A. Blanchard and C. Citti. 2007. Being pathogenic, plastic, and Kluwer Academic/Plenum Publishers, New York, pp. 1–29. sexual while living with a nearly minimal bacterial genome. PLoS. Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Achole- Genet. 3: e75. plasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene Stackebrandt, E. 2009. Class III. Erysipelotrichia. In Bergey’s Manual sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. of Systematic Bacteriology, vol. 3 (edited by de Vos, Garrity, Jones, Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacte- Krieg, Ludwig, Rainey, Schleifer and Whitman). Springer, New York, ria based on comparative sequence analysis of conserved genes. In p. 1298. Microbial Phylogeny and Evolution, Concepts and Controversies Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised (edited by Sapp). Oxford University Press, New York, pp. 70–98. taxonomy of the class Mollicutes - proposed elevation of a mono- Maniloff, J. 2002. Phylogeny and Evolution. In Molecular Biology and phyletic cluster of arthropod-associated mollicutes to ordinal rank Pathogenicity of Mycoplasmas. Kluwer Academic/Plenum Publish- ­( Entomoplasmatales ord. nov.), with provision for familial rank to ers, pp. 31–43. ­separate species with nonhelical morphology (Entomoplasmataceae May, M., R.F. Whitcomb and D.R. Brown. 2009. Mycoplasma and related fam. nov.) from helical species (Spiroplasmataceae), and emended organisms. In CRC Practical Handbook of Microbiology, 2nd edn. descriptions of the order Mycoplasmatales, family Mycoplasmataceae. (edited by Goldman and Green). Taylor & Francis, pp. 456–479. Int. J. Syst. Bacteriol. 43: 378–385. Minion, F.C., E.J. Lefkowitz, M.L. Madsen, B.J. Cleary, S.M. Swartzell Vasconcelos, A.T. and a. coauthors. 2005. Swine and poultry patho- and G.G. Mahairas. 2004. The genome sequence of Mycoplasma hyo- gens: the complete genome sequences of two strains of Mycoplasma pneumoniae strain 232, the agent of swine mycoplasmosis. J. Bacte- hyopneumoniae and a strain of Mycoplasma synoviae. J. Bacteriol. 187: riol. 186: 7123–7133. 5568–5577. Neimark, H.C. 1979. Phylogenetic relationships between mycoplasmas Verbarg, S., H. Rheims, S. Emus, A. Fruhling, R.M. Kroppenstedt, E. and other prokaryotes. In The Mycoplasmas, vol. 1 (edited by Barile Stackebrandt and P. Schumann. 2004. Erysipelothrix inopinata sp. and Razin). Academic Press, New York, pp. 43–61. nov., isolated in the course of sterile filtration of vegetable peptone Oshima, K., S. Kakizawa, H. Nishigawa, H.Y. Jung, W. Wei, S. Suzuki, R. broth, and description of Erysipelotrichaceae fam. nov. Int. J. Syst. Evol. Arashida, D. Nakata, S. Miyata, M. Ugaki and S. Namba. 2004. Reduc- Microbiol. 54: 221–225. tive evolution suggested from the complete genome sequence of a Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Man- plant-pathogenic phytoplasma. Nat. Genet. 36: 27–29. delco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic Papazisi, L., T. Gorton, G. Kutish, P. Markham, G. Browning, D. Nguyen, analysis of the mycoplasmas: basis for their classification. J. Bacteriol. S. Swartzell, A. Madan, G. Mahairas and S. Geary. 2003. The com- 171: 6455–6467. plete genome sequence of the avian pathogen Mycoplasma gallisepti- Westberg, J., A. Persson, A. Holmberg, A. Goesmann, J. Lundeberg, cum strain R(low). Microbiology 149: 2307–2316. K.E. Johansson, B. Pettersson and M. Uhlen. 2004. The genome Pereyre, S., P. Sirand-Pugnet, L. Beven, A. Charron, H. Renaudin, A. sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, Barre, P. Avenaud, D. Jacob, A. Couloux, V. Barbe, A. de Daruvar, the causative agent of contagious bovine pleuropneumonia (CBPP). A. Blanchard and C. Bebear. 2009. Life on arginine for Mycoplasma Genome Res. 14: 221–227. hominis: clues from its minimal genome and comparison with other Woese, C.R., J. Maniloff and L.B. Zablen. 1980. Phylogenetic analysis of human urogenital mycoplasmas. PLoS. Genet. 5: e1000677. the mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 77: 494–498. 574 Phylum XVI. Tenericutes

Order I. Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382

Da n i e l R. Br o w n , Me g h a n Ma y , Ja n e t M. Br a db u r y , Ka r l -Er i k Jo h a n s s o n a n d Ha r o l d Ne i m a rk My.co.plas.ma.ta¢les. N.L. neut. n. Mycoplasma, -atos type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Mycoplasmatales the Mycoplasma order.

The first order in the class Mollicutes is assigned to a group of Pettersson, 2002), the Mycoplasmatales and Entomoplasmatales sterol-requiring, wall-less prokaryotes that occur as commensals represent a clade deeply split from the Acholeplasmatales and or pathogens in a wide range of vertebrate hosts. The descrip- Anaeroplasmatales. tion of the order is essentially the same as for the class. A single A growth requirement for cholesterol or serum is shared by family Mycoplasmataceae with two genera, Mycoplasma and Urea­ the organisms assigned to the order Mycoplasmatales, as well as plasma, recognizes the prominent and distinct characteristics of most other organisms within the class Mollicutes. Therefore, the assigned organisms, based on their sterol requirements for tests for cholesterol requirements are essential to classification. growth, the capacity of some to hydrolyze urea, and conserved Earlier assessments of the growth requirements for cholesterol 16S rRNA gene sequences. were based upon the capacity of organisms to grow in a number Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. of serum-free broth preparations to which various concentra- Comm. Opin. 22, 1958, 166. tions of cholesterol were added (Edward, 1971; Razin and Tully, 1970). In this test, species that do not require exogenous ste- Further descriptive information rol usually show no significant growth response to increasing The entire class Mollicutes was encompassed initially by a single cholesterol concentrations. Polyoxyethylene sorbitan (Tween order. The elevation of acholeplasmas to ordinal rank (Achole­ 80) and palmitic acid should be included in the base medium plasmatales Freundt, Whitcomb, Barile, Razin and Tully 1984) because acholeplasmas such as Acholeplasma axanthum and recognized their major distinctions in nutritional, biochemical, Acholeplasma morum require additional fatty acids for adequate physiological, and genetic characteristics from other members growth. A modified method utilizing serial passage in selective of the class Mollicutes. Subsequently, additional orders were medium has been applied successfully to a large number of proposed to recognize the anaerobic mollicutes and the wall- mollicutes (Rose et al., 1993; Tully, 1995). The Acholeplasmatales less prokaryotes from plants and insects which were phyloge- grow through end-point dilutions in serum-containing medium netically related to the remaining Mycoplasmatales. Thus, the and in serum-free preparations, or occasionally in serum-free Anaeroplasmatales (Robinson and Freundt, 1987) recognized medium supplemented with Tween 80. Mesoplasmas from the the strictly anaerobic, wall-less prokaryotes first isolated from order Entomoplasmatales grow in serum-containing medium and the bovine and ovine rumen, and Entomoplasmatales (Tully in serum-free medium supplemented only with Tween 80. Most et al., 1993) provided a classification for a number of the mol- spiroplasmas, also from the Entomoplasmatales, and all members licutes regularly associated with plant and insect hosts. On the of the order Mycoplasmatales grow only in serum-containing basis of 16S rRNA gene sequence similarities (Johansson and medium.

Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. References J. Bacteriol. 102: 306–310. Edward, D.G. 1971. Determination of sterol requirement for Mycoplas­ Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended clas- matales. J. Gen. Microbiol. 69 : 205–210. sification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. Freundt, E.A. 1955. The classification of the pleuropneumoniae group Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. measuring growth responses of Mollicutes to serum and polyoxyethyl- 5: 67–78. ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised tax- Proposal for elevation of the family Acholeplasmataceae to ordinal onomy of the class Mollicutes - proposed elevation of a monophyletic rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349. cluster of arthropod-associated mollicutes to ordinal rank (Ento­ Johansson, K.E., Pettersson B. 2002. Taxonomy of Mollicutes. In Molecu- moplasmatales ord. nov.), with provision for familial rank to separate lar biology and pathogenicity of mycoplasmas (edited by Razin and species with nonhelical morphology (Entomoplasmataceae fam. nov.) Herrmann). Kluwer Academic, New York, pp. 1–30. from helical species (Spiroplasmataceae), and emended descriptions Judicial Commission. 1958. Opinion 22. Status of the generic name of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bac- Asterococcus and conservation of the generic name Mycoplasma. Int. teriol. 43: 378–385. Bull. Bacteriol. Nomencl. Taxon. 8: 166–168. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sor- Nowak, J. 1929. Morphologie, nature et cycle évolutif du microbe bitan growth requirements of mollicutes. In Molecular and Diagnos- de la péripneumonie des bovidés. Ann. Inst. Pasteur (Paris) 43: tic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). 1330–1352. Academic Press, San Diego, pp. 381–389. Genus I. Mycoplasma 575

Family I. Mycoplasmataceae Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382

Da n i e l R. Br o w n , Me g h a n Ma y , Ja n e t M. Br a db u r y , Ka r l -Er i k Jo h a n s s o n a n d Ha r o l d Ne i m a rk My.co.plas.ma.ta.ce′ae. N.L. neut. n. Mycoplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Mycoplasmataceae the Mycoplasma family. Pleomorphic usually coccoid cells, 300–800 nm in diameter, to Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. slender branched filaments of uniform diameter. Some cells Comm. Opin. 22, 1958, 166. have a terminal bleb or tip structure that mediates adhesion to Further descriptive information certain surfaces. Cells lack a cell wall and are bounded only by a plasma membrane. Gram-stain-negative due to the absence of This family and its type genus Mycoplasma are polyphyletic. Two a cell wall. Usually nonmotile. Facultatively anaerobic in most genera, Mycoplasma and Ureaplasma, are currently accepted instances, possessing a truncated flavin-terminated electron within the family. The genus Mycoplasma is further divisible into transport chain devoid of quinones and cytochromes. Colonies phylogenetic groups on the basis of 16S rRNA gene sequence of Mycoplasma are usually less than l mm in diameter and colo- similarities (Johansson and Pettersson, 2002), including an eco- nies of Ureaplasma are much smaller than that. The typical col- logically, phenotypically, and genetically cohesive group called ony has a fried-egg or “cauliflower head” appearance. Usually the mycoides cluster, which includes the type species Mycoplasma catalase-negative. Chemo-organotrophic, usually using either mycoides and other major pathogens of ruminant animals. The sugars or arginine, but sometimes both, or having an obligate taxonomic position of the mycoides cluster is an important requirement for urea as the major energy source. Require cho- anomaly because molecular markers based upon rRNA and lesterol or related sterols for growth. Commensals or pathogens other gene sequences indicate that it is closely related to other of a wide range of vertebrate hosts. The genome size ranges genera usually associated with plant and insect hosts and cur- from about 580 to 1350 kbp, as measured by pulsed field gel rently classified within the order Entomoplasmatales. Members of electrophoresis (PFGE) or complete DNA sequencing. the genus Ureaplasma are distinguished by their tiny colony size

DNA G+C content (mol%): about 23–40 (Bd, Tm). and ­capacity to hydrolyze urea.

Genus I. Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166AL

Da n i e l R. Br o w n , Me g h a n Ma y , Ja n e t M. Br a db u r y , Mi t c h e l l F. Ba l i s h , Mi c h a e l J. Ca l c u t t , Jo h n I. Gl a s s , Sé v e r i n e Ta s k e r , Jo a n n e B. Me s s i c k , Ka r l -Er i k Jo h a n s s o n a n d Ha r o l d Ne i m a rk My.co.plas¢ma. Gr. masc. n. myces a fungus; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Mycoplasma fungus form.

Pleomorphic cells, 300–800 nm in diameter, varying in shape culture medium, and the growth phase. Some mycoplasmas from spherical, ovoid or flask-shaped, or twisted rods, to slender are filamentous in their early and exponential growth phases branched filaments ranging in length from 50 to 500 nm. Cells or when attached to surfaces or other cells. This form can lack a cell wall and are bounded by a single plasma membrane. be transitory, and the filaments may branch or fragment into Gram-stain-negative due to the absence of a cell wall. Some have chains of cocci or individual vegetative cells. Many species are a complex internal cytoskeleton. Some have a specific tip struc- typically coccoid and never develop a filamentous phase. Some ture that mediates attachment to host cells or other surfaces. species develop specialized attachment tip structures involved Usually nonmotile, but gliding motility has been demonstrated in colonization and virulence (Figure 106). In Romanowsky- in some species. Aerobic or facultatively anaerobic. Optimum type stained blood smears, hemotropic species (trivial name, growth at 37°C is common, but permissive growth temperatures hemoplasmas) appear as round to oval cells on the surface of range from 20 to 45°C. Chemo-organotrophic, usually using erythrocytes (Figure 107). They may be found individually or, either sugars or arginine as the major energy source. Require during periods of high parasitemia, in pairs or chains giving the cholesterol or related sterols for growth. Colonies are usually appearance of pleomorphism. Their small size and the absence less than l mm in diameter. The typical colony has a fried-egg of cell wall components provide considerable plasticity to appearance. The genome size of species examined ranges from the organisms, so that cells of most species are readily filterable 580 kbp to about 1350 kbp. The codon UGA encodes trypto- through 450 nm pores, and many species have some cells in the phan in all species examined. Commensals or pathogens in a population that are able to pass through 220 nm or even 100 wide range of vertebrate hosts. nm filters (Tully, 1983). Descriptions of the morphology, ultra- DNA G+C content (mol%): 23–40. structure, and motility of mycoplasmas should be based on cor- Type species: Mycoplasma mycoides (Borrel, Dujardin- relation of the appearance of young exponential-phase broth ­Beaumetz, Jeantet and Jouan 1910) Freundt 1955, 73 (Astero- cultures under phase-contrast or dark-field microscopy with coccus mycoides Borrel, Dujardin-Beaumetz, Jeantet and Jouan their appearance using negative-staining or electron micros- 1910, 179). copy (Biberfeld and Biberfeld, 1970; Boatman, 1979; Carson et al., 1992; Cole, 1983). Special attention to the osmolarity of Further descriptive information the fixatives and buffers is required since these may alter the The shape of these organisms (trivial name, mycoplasmas) size and shape of the organisms. The classical isolated colony can depend on the osmotic pressure, nutritional quality of the is umbonate with a fried-egg appearance, but others may have 576 Family I. Mycoplasmataceae

Figure 106. Diverse cellular morphology in the genus Mycoplasma. Scanning electron micrographs of cells of (a) Mycoplasma penetrans, (b) Mycoplasma pneumoniae, (c) “Mycoplasma insons”, and (d) Mycoplasma genitalium. Bar = 1 mm. Images provided by Dominika Jurkovic, Jennifer Hatchel, Ryan Relich and Mitchell Balish.

Figure 107. Hemotropic mycoplasmas. (a) Scanning electron micrograph of Mycoplasma ovis cells colonizing the surface of an erythrocyte (Neimark et al., 2004); bar = 500 nm. (b) Transmission electron micrograph showing fibrils bridging the space between a “Candidatus Mycoplasma kahaneii” cell and a depression in the surface of a colonized erythrocyte (Neimark et al., 2002a); bar = 250 nm. Images used with permission. either cauliflower-like or smooth colony surfaces (Figure 108), involved in morphogenesis, motility, cytadherence, and cell divi- with smooth, irregular or scalloped margins, depending on the sion (Balish and Krause, 2006). In the distantly related species species, agar concentration, and other growth conditions. Mycoplasma pneumoniae and Mycoplasma mobile, the cytoskeleton A significant minority of species exhibit cell polarization. underlies a terminal organelle. This prominent extension of the This depends on Triton X-100-insoluble cytoskeletal structures cytoplasm and cell membrane is the principal focus of adherence Genus I. Mycoplasma 577

Figure 108. Diverse colonial morphology in the genus Mycoplasma. (a) Mycoplasma mycoides PG1T (diameter 0.50–0.75 mm), (b) Mycoplasma hyopneumoniae NCTC 10110T (diameter 0.15–0.20 mm), (c) Mycoplasma pneumoniae NCTC 10119T (diameter 0.05–0.10 mm), and (d) Mycoplasma hyorhinis ATCC 29052 (diameter 0.25–0.30 mm) after 3, 7, 5, and 6 d growth, respectively, on Mycoplasma Experience Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification 25×. Images provided by Helena Windsor and David Windsor. and is the leading end of cells engaged in gliding motility. In found in Mycoplasma pneumoniae, suggesting that it has evolved Mycoplasma pneumoniae, adhesin proteins are located either all independently. Further distinct cytoskeletal structures appear in over the surface of the organelle or at its distal tip; an unrelated Mycoplasma penetrans (Jurkovic and Balish, unpublished), “Myco- adhesin is concentrated at the base of the terminal organelle plasma insons” (Relich et al., 2009), and several species of the in Mycoplasma mobile (Balish, 2006). Both the formation of this mycoides cluster (Peterson et al., 1973). attachment organelle of Mycoplasma pneumoniae and the local- Attachment to eukaryotic host cells is important for the natu- ization of the adhesins depend upon cytoskeletal proteins that ral survival and transmission of mycoplasmas. The prominent form an electron-dense core within its cytoplasm, which is sur- attachment organelle of species in the Mycoplasma pneumoniae rounded by an electron-lucent space (Krause and Balish, 2004). cluster is the most extensively characterized determinant of cytad- The overall appearance of this core is that of two parallel, flat herence. In other species, multiple adhesin proteins are involved rods of differing thickness, with a bend near the cell-proximal in cytadherence. When one adhesin is blocked, cytadherence is end (Henderson and Jensen, 2006; Seybert et al., 2006). A bi- reduced, but not completely lost. For this reason, the adhesins lobed button constitutes its distal end and its proximal base ter- appear to be functionally redundant rather than synergistic in minates in a bowl-like structure. Overall, both the core and the action. Numerous species possess multigene families of antigeni- attachment organelle are 270–300 nm in length (Hatchel and cally variable proteins, some of which have been implicated in Balish, 2008). Around the onset of DNA replication, a second host cell attachment or hemagglutination. While this attach- attachment organelle is constructed (Seto et al., 2001). The ment may serve as a supplemental binding mechanism in species motile force provided by the first organelle reorganizes the cell such as Mycoplasma gallisepticum and Mycoplasma hominis, variable such that the new organelle is moved to the opposite cell pole surface proteins are currently the only known mechanism for before cell division (Hasselbring et al., 2006). These observa- cytadherence and hemagglutination of Mycoplasma synoviae and tions suggest that complex coordination exists between attach- Mycoplasma pulmonis. The avidity of adherence may differ among ment organelle biogenesis, motor activity, the DNA replication variants in Mycoplasma pulmonis and Mycoplasma hominis. Though machinery, and the cytokinetic machinery. Similar structures are one or more attachment mechanisms have been described for present in other species of the Mycoplasma pneumoniae cluster, but numerous species, there remains a greater number of species in most cases the attachment organelle is shorter, resulting in with no documented system for cytadherence. Strains that lose much of the core protruding into the cell body (Hatchel and Bal- the capacity to cytadhere are almost invariably unable to survive ish, 2008). In Mycoplasma mobile, the terminal organelle is com- in their hosts, but highly invasive species such as Mycoplasma pletely dissimilar, consisting of a cell-distal sphere with numerous ­alligatoris may not require host cell attachment for infection. tentacle-like strands extending into the cytoplasm (Nakane and The mycoplasmas possess a typical prokaryotic plasma Miyata, 2007). It is comprised of proteins unrelated to those ­membrane composed of amphipathic lipids and proteins 578 Family I. Mycoplasmataceae

­(McElhaney, 1992a, b, c; Smith, 1992; Wieslander et al., 1992). ­arginine-hydrolyzing species can be enhanced by supplementing At one time, demonstration of a single unit membrane was media with arginine. Commonly used alternatives such as Frey’s, mandatory for defining all novel species of mollicutes (Tully, Hayflick’s and Friis’ media differ from SP-4 mainly in the propor- 1995a). Now, when the 16S rRNA gene sequence of a novel spe- tions of inorganic salts, amino acids, serum sources, and types cies is determined and the candidate is placed in one of the phy- of antibiotics. For species that utilize both sugars and arginine logenetic clusters of mollicutes, in the majority of cases it can as carbon sources, the pH of the medium may initially decrease be safely inferred that the organism lacks a cell wall, because before rising later during the course of growth (Razin et al., the majority of others in that cluster will have been shown to 1998). Defined mycoplasma culture media have been described be solely membrane-bound (Brown et al., 2007). The lack of in detail (Rodwell, 1983), but provision of lipids and amino acids a cell wall explains the resistance of the organisms to lysis by in the appropriate ratios is difficult technically (Miles, 1992b). lysozyme and their susceptibility to lysis by osmotic shock and Many mobile genetic elements occur in the genus. Four plas- various agents causing the lysis of bacterial protoplasts (Razin, mids have been identified in members of the mycoides clus- 1979, 1983). In certain species, the extracellular surface is tex- ter (Bergemann and Finch, 1988; Djordjevic et al., 2001; King tured with capsular material or a nap, which can be stained with and Dybvig, 1994). Each plasmid is apparently cryptic, with no ruthenium red in some cases (Rosenbusch and Minion, 1992). discernible determinants for virulence or antibiotic resistance. These organisms represent some of the most nutritionally DNA viruses have been isolated from Mycoplasma bovirhinis fastidious prokaryotes, as expected from their greatly reduced (Howard et al., 1980), Mycoplasma hyorhinis (Gourlay et al., or minimalist genomes, close association with vertebrate hosts 1983) Mycoplasma pulmonis (Tu et al., 2001), and Mycoplasma as commensals and pathogens, and total dependence upon the arthritidis (Voelker and Dybvig, 1999). The Mycoplasma pulmonis host to meet all nutritional requirements. They have very limited P1 virus and the lysogenic bacteriophage MAV1 of Mycoplasma capacity for intermediary metabolism, which restricts the utility of arthritidis do not share sequence similarity (Tu et al., 2001; conventional biochemical tests for identification. Detailed infor- Voelker and Dybvig, 1999), whereas the Mycoplasma fermentans mation on carbohydrate (Pollack, 1992, 1997, 2002; Pollack et al., MFV1 prophage is strikingly similar in genetic organization to 1996), lipid (McElhaney, 1992a), and amino acid (Fischer et al., MAV1 (Röske et al., 2004). No role in pathobiology has been 1992) metabolism is available. All species examined have trun- demonstrated for any virus or prophage. cated respiratory systems, lack a complete tricarboxylic acid cycle, The most abundant mobile DNAs in Mycoplasma are inser- and lack quinones or cytochromes, which precludes their capacity tion sequence (IS) elements. The first identified units (IS1138 to carry out oxidative phosphorylation. Instead only low levels of of Mycoplasma pulmonis, IS1221 of Mycoplasma hyorhinis, IS1296 ATP may be generated through glycolysis or the arginine dihy- of Mycoplasma mycoides subsp. mycoides and ISMi1 of Mycoplasma drolase pathway (Miles, 1992a, b). Fermentative species catabo- fermentans) are members of the IS3 family (Bhugra and Dyb- lize glucose or other carbohydrates to produce ATP and acid and, vig, 1993; Ferrell et al., 1989; Frey et al., 1995; Hu et al., 1990). consequently, lower the pH of the medium. Non-fermentative spe- More recently, multiple IS elements of divergent subgroups have cies hydrolyze arginine to yield ammonia, some ATP, and carbon been identified. Members of the IS4 family include IS1634 and dioxide, and consequently raise the pH of the medium. Species ISMmy1 of Mycoplasma mycoides subsp. mycoides (Vilei et al., 1999; such as Mycoplasma fermentans have both pathways. Species such as Westberg et al., 2002), ISMhp1 of Mycoplasma hyopneumoniae, Mycoplasma bovis evidently lack both pathways, but are capable oxi- ISMhp1-like unit of Mycoplasma synoviae, and four distinct ele- dizing pyruvate or lactate to yield ATP (Miles, 1992a; Taylor et al., ments of Mycoplasma bovis (Lysnyansky et al., 2009). Among the 1994). Some species cause a pronounced “film and spots” reac- IS30 family members identified are IS1630 of Mycoplasma fermen- tion on media incorporating heat-inactivated horse serum or egg tans, ISMhom1 from Mycoplasma hominis, ISMag1 of Mycoplasma yolk: a wrinkled film composed of cholesterol and phospholipids agalactiae (Pilo et al., 2003), and two IS units of Mycoplasma bovis. forms on the surface of the medium and dark spots containing IS-like elements have also been identified in Mycoplasma leachii, salts of fatty acids appear around the colonies. Mycoplasma penetrans (belonging to four different families), Myco- Most mycoplasmas are aerobes or facultative anaerobes, but plasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma orale. some species such as Mycoplasma muris prefer an anaerobic Transposases that reside within IS units are also discernable in environment. The optimum growth of species isolated from the genome of Mycoplasma gallisepticum (Papazisi et al., 2003). homeothermic hosts is commonly at 37°C and the permissive In select instances, almost identical IS units have been found temperature range of species from poikilothermic fish and in species from different phylogenetic clades, which strongly reptiles is always above 20–25°C. Thus, growth of the myco- suggests lateral gene transfer between species. Despite their plasmas is restricted to mesophilic temperatures. Growth in widespread distribution, IS elements are not ubiquitous in the liquid cultures usually produces at most light turbidity and few genus. Although the type strains of Mycoplasma bovis (54 IS units sedimented cells, except for the heavy turbidity and sediments of seven different types) and Mycoplasma mycoides subsp. mycoides usually observed with members of the Mycoplasma mycoides clus- (97 elements of three different types) possess large numbers of ter. Tully (1995b) described in detail the most commonly used elements, the sequenced genomes of Mycoplasma arthritidis, Myco- culture media formulations. Although colonies are occasionally plasma genitalium, Mycoplasma pneumoniae, and Mycoplasma mobile first detected on blood agar, complex undefined media such as lack detectable IS units. American Type Culture Collection (ATCC) medium 988 (SP-4) Although IS units only encode genes related to transposition, are usually required for primary isolation and maintenance. large integrating elements have also been identified in diverse Cell-wall-targeting antibiotics are included to discourage Mycoplasma species. The Integrative Conjugal Elements (ICE) growth of other bacteria. Phenol red facilitates detection of of Mycoplasma fermentans strain PG18T comprise >8% of the species that excrete acidic or alkaline metabolites. Growth of genome and related units have been identified in Mycoplasma Genus I. Mycoplasma 579 agalactiae, Mycoplasma bovis, Mycoplasma capricolum, Mycoplasma types of Mycoplasma mycoides subsp. mycoides, and Mycoplasma hyopneumoniae, and Mycoplasma mycoides subsp. mycoides. In gen- bovis. Mycoplasma mycoides subsp. mycoides has caused major losses eral, such units encode 18–30 genes, can be detected in extra- of livestock globally in the twentieth century due to contagious chromosomal forms, and are strain-variable in distribution and bovine pleuropneumonia and currently remains a problem in chromosomal insertion site. Two additional large mobile DNAs, Asia and Africa (Lesnoff et al., 2004). Mycoplasma bovis is a wide- designated Tra Islands, were identified in Mycoplasma capricolum spread agent of otitis media, pneumonia, mastitis, polyarthri- California kidT. The presence of putative conjugation genes tis, and urogenital disease in cattle and buffaloes. Mycoplasma and the variability in genomic location of Tra Islands and ICE mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum, and suggest that these are agents of lateral gene transfer. Mycoplasma agalactiae are important causes of arthritis, mastitis, The best-studied mycoplasmas are primary pathogens of and agalactia in goats and sheep. Mycoplasma mycoides subsp. capri humans or domesticated animals (Baseman and Tully, 1997). (type strain PG3T) properly includes all of the serovars histori- About half of the listed species occur in the absence of disease, cally called “Large Colony” types of subspecies mycoides (Manso- but are occasional opportunistic or secondary pathogens. The Silván et al., 2009; Shahram et al., 2010). Mycoplasma capricolum principal human pathogens are Mycoplasma pneumoniae, Myco- subsp. capripneumoniae (type strain F38T) causes severe conta- plasma hominis, and Mycoplasma genitalium, with Mycoplasma pen- gious pleuropneumonia in goats (Leach et al., 1993; McMartin etrans added to this list due to its association with HIV infections et al., 1980). Contagious bovine and caprine pleuropneumonia, (Blanchard, 1997; Blanchard et al., 1997; Tully, 1993; Waites and and mycoplasmal agalactia of sheep or goats are subject to con- Talkington, 2005). Mycoplasma pneumoniae is one of the main trol through listing in the Terrestrial Animal Health Code of the agents of community-acquired pneumonia, bronchitis, and Office International des Epizooties (http://oie.int) as well as other respiratory complications (Atkinson et al., 2008). Myco- strict notification and export regulations by individual countries. plasma pneumoniae infections can also involve extra-pulmonary Mycoplasma hyopneumoniae, one of the most difficult species to complications including central nervous system, cardiovascular, cultivate, causes primary enzootic pneumonia in pigs and exac- and dermatological manifestations. Outbreaks cause consider- erbates other porcine respiratory diseases leading to substantial able morbidity and require rapid and effective therapeutic inter- economic burdens. Mycoplasma hyosynoviae is carried in the upper vention (Hyde et al., 2001; Meyer and Clough, 1993). Mycoplasma respiratory tract, but causes nonsuppurative polyarthritis, usually hominis occurs more frequently in the urogenital tract of women without other serositis, especially in growing pigs. than men and is often found in the genital tract of women with The most important poultry pathogens are Mycoplasma gal- vaginitis, bacterial vaginosis, or localized intrauterine infections lisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis, but (Keane et al., 2000). The organism can gain access to a fetus more than 20 other species have been isolated from birds as from uterine sites and it is associated with perinatal morbidity diverse as ostriches, raptors, and penguins (Bradbury and and mortality (Gonçalves et al., 2002; Waites et al., 1988). It is ­Morrow, 2008). Mycoplasma gallisepticum can be transmitted ver- also clearly associated with septicemias and respiratory infections tically, venereally, by other direct contact, or by aerosol to cause and with transplant or joint infections in immunosuppressed respiratory disease and its sequelae in chickens, turkeys, and persons (Brunner et al., 2000; Busch et al., 2000; Fernandez other birds. It also causes decreased egg production and egg Guerrero et al., 1999; Garcia-Porrua et al., 1997; Gass et al., quality in chickens. Mycoplasma synoviae can cause a syndrome 1996; Hopkins et al., 2002; Mattila et al., 1999; Tully, 1993; Zheng of synovitis, tendonitis, and bursitis in addition to respiratory et al., 1997). Mycoplasma genitalium has been associated with non- disease in chickens and turkeys, whereas the developmental gonococcal urethritis in men (Gambini et al., 2000; Jensen, 2004; abnormalities and airsacculitis associated with congenital or Jensen et al., 2004; Taylor-Robinson et al., 2004; Taylor-Robinson acquired Mycoplasma meleagridis infection seem restricted to tur- and Horner, 2001; Totten et al., 2001) and urogenital disease in keys. Mycoplasma gallisepticum and Mycoplasma synoviae are also women (Baseman et al., 2004; Blaylock et al., 2004). Mycoplasma listed in the OIE’s Terrestrial Animal Health Code. genitalium occurs more frequently in the vagina than in the cer- Pathogenicity of specific mycoplasmas has also been reported vix or urethra, but it may be involved in cervicitis (Casin et al., for companion animals (Chalker, 2005; Lemcke, 1979; ­Messick, 2002; Manhart et al., 2001). Mycoplasmas are common agents 2003) and a number of wild animal hosts (Brown et al., 2005). of chronic joint inflammation in a wide variety of hosts (Cole The respiratory, reproductive, and joint diseases caused in et al., 1985). Species associated with arthritis in humans include rodents by Mycoplasma pulmonis and Mycoplasma arthritidis Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium, (Schoeb, 2000) are important models of infection and immu- Mycoplasma salivarium, and possibly Mycoplasma pneumoniae in nity in humans and other animals. juvenile arthritis (Waites and Talkington, 2005). Humans are also Hemoplasmas infect a variety of wild and domesticated susceptible to opportunistic zoonotic mycoplasmosis; immuno- ­animals and are relatively host-specific, although cross-­infection suppressed persons are highly susceptible. The recent molecular of related hosts has been reported. Transmission can be achieved confirmation of a Mycoplasma haemofelis-like infection in an HIV- by ingestion of infected blood or by percutaneous inoculation. positive patient (dos Santos et al., 2008) highlights the zoonotic Arthropod vector transmission of some species is also supported potential of the hemoplasmas. by experimentation and by the ­clustered ­geographic distribu- Mycoplasmas colonize fish, reptiles, birds, and terrestrial tion of hemoplasmosis in some studies (Sykes et al., 2007; Willi and aquatic mammals. Some cause significant diseases of cat- et al., 2006a). The pathogenicity of different hemoplasma spe- tle and other ruminants, swine, poultry, or wildlife, and others cies is variable and strain virulence also likely plays a key role in are opportunistic or secondary veterinary pathogens (Simecka the development of disease. For example, Mycoplasma haemofelis et al., 1992; Tully and Whitcomb, 1979). The principal bovine can induce acute clinical disease in non-splenectomized, immu- pathogens include the serovars historically called “Small Colony” nocompetent cats, whereas Mycoplasma haemocanis appears able 580 Family I. Mycoplasmataceae to induce disease only in immunosuppressed or splenectomized Because they lack lipopolysaccharide and a cell wall, and do dogs. Clinical syndromes range from acute fatal hemolytic ane- not synthesize their own nucleotides, mycoplasmas are intrin- mia to chronic insidious anemia and ill-thrift. Signs may include sically resistant to polymixins, b-lactams, vancomycin, fosfomy- anemia, pyrexia, anorexia, dehydration, weight loss, and infer- cin, sulfonamides, and trimethoprim. They are also resistant to tility. The presence of erythrocyte-bound antibodies (including rifampin because their RNA polymerase is not affected by that cold agglutinins), indicated by positive Coombs’ testing, has antibiotic (Bébéar and Kempf, 2005). Individual species exhibit been demonstrated in some hemoplasma-infected animals and an even broader spectrum of antibiotic resistance, such as the may contribute to anemia. Animals can remain chronic asymp- resistance to erythromycin and azithromycin exhibited by sev- tomatic carriers of hemoplasmas after acute infection. PCR is the eral species, which is apparently mediated by mutation in the diagnostic test of choice for hemoplasma infection. 23S rRNA (Pereyre et al., 2002). Treatment of mycoplasmosis Contamination of eukaryotic cell cultures with mollicutes is still often involves the use of antibiotics that inhibit protein syn- a common and important yet often unrecognized problem (Tully thesis or DNA replication. Certain macrolides or ketolides are and Razin, 1996). More than 20 species have been isolated from used when tetracyclines or fluoroquinolones are inappropri- contaminated cell lines, but more than 90% of the contamination ate. Fluoroquinolones, aminoglycosides, pleuromutilins, and is thought to be caused by just five species of mycoplasma: Myco- phenicols are not widely used to treat human mycoplasmosis plasma arginini, Mycoplasma fermentans, Mycoplasma hominis, Myco- at present, with the exception of chloramphenicol for neonates plasma hyorhinis, and Mycoplasma orale, plus Acholeplasma laidlawii. with mycoplasmosis of the central nervous system unresponsive Mycoplasma pirum and Mycoplasma salivarium account for most of to other antibiotics (Waites et al., 1992), but their use in veteri- the remainder (Drexler and Uphoff, 2002). Culture medium com- nary medicine is more common. The long-term antimicrobial ponents of animal origin, passage of contaminated cultures, and therapy often required may be due to mycoplasmal sequestra- laboratory personnel are likely to be the most significant sources tion in privileged sites, potentially including inside host cells. of cell culture contaminants. PCR-based approaches to detection Mycoplasmosis in immunodeficient patients is very difficult to achieve sensitivity and specificity far superior to fluorescent stain- control with antibiotic drugs (Baseman and Tully, 1997). ing methods (Masover and Becker, 1996). Another method of Enrichment and isolation procedures detection is based on mycoplasma-specific ATP synthesis activity present in contaminated culture medium (Robertson and Stemke, Techniques for isolation of mycoplasmas from humans, various 1995; MycoAlert, Lonza Group). Eradication through treatment of species of animals, and from cell cultures have been described contaminated cultures with antibiotics (Del Giudice and Gardella, (Neimark et al., 2001; Tully and Razin, 1983). Typical steps in the 1996) is rarely successful. Strategies for prevention and control of isolation of mycoplasmas were outlined in the recently revised mycoplasmal contamination of cell cultures have been described minimal standards for descriptions of new species (Brown et al., in detail (Smith and Mowles, 1996). 2007). Initial isolates may contain a mixture of species, so cloning Several categories of potential virulence determinants are by repeated filtration through membrane filters with a pore size encoded in the metagenome of pathogenic mycoplasmas. Some of 450 or 220 nm is essential. The initial filtrate and dilutions of species possess multiple types of virulence factors. Determinants it are cultured on solid medium and an isolated colony is subse- such as adhesins and accessory proteins, extracellular polysaccha- quently picked from a plate on which only a few colonies have ride structures, and pro-inflammatory or pro-­apoptotic membrane developed. This colony is used to found a new cultural line, which lipoproteins are produced by multiple species. Several species is then expanded, filtered, plated, and picked two additional times. excrete potentially toxic by-products of intermediary metabolism, Hemoplasmas have not yet been successfully grown in continuous including hydrogen peroxide, superoxide radicals, or ammonia. culture in vitro, although recent work (Li et al., 2008) suggests that Other determinants such as extracellular endopeptidases, nucle- in vitro maintenance of Mycoplasma suis may be possible. ases, and glycosidases seem irregularly distributed in the genus, Maintenance procedures whereas the ADP-ribosylating and vacuolating cytotoxin (pertus- sis exotoxin S1 subunit analog) of Mycoplasma pneumoniae and the Cultures of mycoplasma can be preserved by lyophilization or cryo- T-lymphocyte mitogen (superantigen) of Mycoplasma arthritidis are genic storage (Leach, 1983). The serum in the culture medium evidently unique to those species. Reports of a putative exotoxin provides effective cryoprotection, but addition of sucrose may elaborated by Mycoplasma neurolyticum have not been substantiated enhance survival following lyophilization. Hemoplasmas can be by later work (Tryon and Baseman, 1992). frozen in heparin- or EDTA-anticoagulated blood cryopreserved Candidate virulence mechanisms, such as motility, biofilm with dimethylsulfoxide. Most species can be recovered with little formation, or facultative intracellular invasion, are expressed loss of viability even after storage for many years. by a range of pathogenic species. Several species possess Taxonomic comments systems of variable surface antigens that are thought to be important in evasion of the hosts’ adaptive immune responses. This polyphyletic genus is divisible on the basis of 16S rRNA and In addition, a large number of species can suppress or inappro- other gene sequence similarities into a large paraphyletic clade priately stimulate host immune cells and their receptors and of over 100 species in two groups called hominis and pneumo- cytokines through diverse, poorly characterized mycoplasmal niae (Johansson and Pettersson, 2002; Figure 109), plus the components. Although candidate virulence factor discovery has ecologically, phenotypically, and genetically cohesive “mycoides accelerated significantly in recent years through whole genome cluster” of five species including the type species Mycoplasma annotation, the molecular basis for pathogenicity and causal mycoides (Cottew et al., 1987; Manso-Silván et al., 2009; ­Shahram relationships with disease still remain to be definitively estab- et al., 2010). The priority of Mycoplasma mycoides as the type spe- lished for most of these factors (Razin and Herrmann, 2002; cies of the genus Mycoplasma and, hence, the family Mycoplasmata- Razin et al., 1998). ceae and the order Mycoplasmatales is, in retrospect, unfortunate. Genus I. Mycoplasma 581

The phylogenetic position of the mycoides cluster is eccentrically (Lartigue et al., 2007). Cloning in yeast and subsequent resur- situated to the remaining species of the order Mycoplasmatales, rection of Mycoplasma mycoides “Large Colony” genomes as living amidst genera that are properly classified in the order Entomoplas- bacteria demonstrate that it is possible to enliven a prokaryotic matales. When the order Entomoplasmatales was established, a cen- genome constructed in a eukaryotic cell (Lartigue et al., 2009). tury after the discovery of Mycoplasma mycoides, it was explicitly The ICSP subcommittee on the taxonomy of Mollicutes may be accepted that the taxonomic anomaly created by the phyloge- the first to accommodate a system of nomenclature and classifi- netic position of the mycoides cluster will remain impractical to cation for species of novel prokaryotes that originate by entirely resolve (Tully et al., 1993). The few species in the mycoides cluster artificial speciation events (Brown and Bradbury., 2008). cannot simply be renamed, because confusion and peril would result, especially regarding “Small Colony” PG1T-like strains of Differentiation of the genus Mycoplasma Mycoplasma mycoides subsp. mycoides and F38T-like strains of Myco- from other genera plasma capricolum subsp. capripneumoniae, which are highly virulent Properties that partially fulfill criteria for assignment to the pathogens and subject to strict international regulations. class Mollicutes (Brown et al., 2007) include absence of a cell Another controversy involves the nomenclature of uncultivated wall, filterability, and the presence of conserved 16S rRNA gene hemotropic bacteria originally assigned to the genera Eperythro- sequences. Aerobic or facultatively anaerobic growth in artificial zoon or Haemobartonella. It is now undisputed that, on the bases of medium and a growth requirement for sterols exclude assign- their lack of a cell wall, small cell size, low G+C content, use of ment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, the codon UGA to encode tryptophan, regular association with or “Candidatus Phytoplasma”. Non-spiral cellular morphology vertebrate hosts, and 16S rRNA gene sequences that are most simi- and regular association with a vertebrate host or fluids of ver- lar (80–84%) to species in the pneumoniae group of Mycoplasma, tebrate origin support exclusion from the genera Spiroplasma, these organisms are properly affiliated with the Mycoplasmatales. Entomoplasma, or Mesoplasma. The inability to hydrolyze urea However, the proposed transfers of Eperythrozoon and Haemobarto- excludes assignment to the genus Ureaplasma. nella species to the genus Mycoplasma (Neimark et al., 2001, 2005) were opposed on the grounds that the degree of 16S rRNA gene Acknowledgements sequence similarity is insufficient (Uilenberg et al., 2004, 2006). The principal objection to establishing the hemoplasmas in a third The lifetime achievements in mycoplasmology and substantial genus in the Mycoplasmataceae­ ­(Uilenberg et al., 2006) is that this contributions to the preparation of this material by Joseph G. would compound the polyphyly within the pneumoniae group Tully are gratefully acknowledged. Daniel R. Brown and Meghan solely on the basis of a capacity to adhere to erythrocytes in vivo. In May were supported by NIH grant 5R01GM076584. Séverine addition, the transfer of the type species Eperythrozoon coccoides to Tasker was supported by Wellcome Trust grant WT077718. the genus Mycoplasma is complicated by priority because Eperythro- Further reading zoon predates Mycoplasma. The alternative, to transfer all mycoplas- mas to the genus Eperythrozoon, would be completely impractical Blanchard, A. and G. Browning (editors). 2005. Mycoplasmas: and perilous in part because the epithet Eperythrozoon does not Molecular Biology, Pathogenicity, and Strategies for Control. indicate an affiliation with Mycoplasmatales. The Judicial Commis- Horizon Press, Norwich, UK. sion of the International Committee on Systematics of Prokaryotes Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (edi- (ICSP) declined to rule on a request for an opinion in this matter tors). 1992. Mycoplasmas: Molecular Biology and Pathogen- (Neimark et al., 2005) during their 2008 meeting, but a provisional esis. American Society for Microbiology, Washington, D.C. placement in the genus Mycoplasma has otherwise been embraced Razin, S. and J.G. Tully (editors). 1995. Molecular and Diagnos- by specialists in the molecular biology and clinical pathogenicity of tic ­Procedures in Mycoplasmology, vol. 1, Molecular Charac- these and similar hemotropic organisms. At present, the designa- terization. Academic Press, San Diego. tion “Candidatus” must still be used for new types. Tully, J.G. and S. Razin (editors). 1996. Molecular and Diagnos- Mycoplasma feliminutum was first described during a time when tic Procedures in Mycoplasmology, vol. 2, Diagnostic Proce- the only named genus of mollicutes was Mycoplasma. Its publication dures. Academic Press, San Diego. coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is Differentiation of the species of the genus Mycoplasma properly affiliated through established phenotypic (Heyward et al., Glucose fermentation and arginine hydrolysis are discriminat- 1969) and 16S rRNA gene sequence (Brown et al., 1995) similari- ing phenotypic markers (Table 137), but the pleomorphism and ties. This explains the apparent inconsistencies with its assignment metabolic simplicity of mycoplasmas has led to a current reliance to the genus Mycoplasma. The name Mycoplasma feliminutum should principally on the combination of 16S rRNA gene sequencing and therefore be revised to Acholeplasma feliminutum comb. nov. The reciprocal serology for species differentiation. Failure to cross- type strain is BenT (=ATCC 25749T; Heyward et al., 1969). react with antisera against previously recognized species provides Recent work at the J. Craig Venter Institute, including com- substantial evidence for species novelty. For this reason, deposi- plete chemical synthesis and cloning of an intact Mycoplasma tion of antiserum against a novel type strain into a recognized genitalium chromosome (Gibson et al., 2008) and other work collection is still mandatory for novel species descriptions (Brown with Mycoplasma mycoides “Large Colony” (Lartigue et al., 2009), et al., 2007). Preliminary differentiation can be by PCR and DNA suggests that de novo synthesis of two species of mollicute is immi- sequencing using primers specific for bacterial 16S rRNA genes nent. Transplantation of isolated deproteinized tetR-selectable or the 16S–23S intergenic region. A similarity matrix relating the chromosomes from donor Mycoplasma mycoides “Large Colony” candidate strain to its closest neighbors, usually species with >94% into recipient Mycoplasma capricolum cells displaced the recipient 16S rRNA gene sequence similarity, will suggest related species genome and conferred the genotype and phenotype of the donor that should be examined for serological cross-reactivities. 582 Family I. Mycoplasmataceae

Mycoplasma equigenitalium Mycoplasma elephantis equigenitalium cluster Mycoplasma bovis Mycoplasma agalactiae * Mycoplasma primatum Mycoplasma opalescens Mycoplasma spermatophilum Mycoplasma fermentans * Mycoplasma caviae Mycoplasma adleri * * Mycoplasma felifaucium * Mycoplasma leopharyngis Mycoplasma maculosum bovis cluster Mycoplasma lipofaciens Mycoplasma bovigenitalium ** Mycoplasma californicum Mycoplasma simbae Mycoplasma phocirhinis Mycoplasma meleagridis * Mycoplasma gallinarum Mycoplasma iners * Mycoplasma columbinasale Mycoplasma columbinum * Mycoplasma lipophilum Mycoplasma hyopharyngis lipophilum cluster Mycoplasma sphenisci Mycoplasma synoviae Mycoplasma verecundum Mycoplasma gallinaceum Mycoplasma corogypsi Mycoplasma glycophilum Mycoplasma gallopavonis Mycoplasma buteonis * Mycoplasma felis * * * Mycoplasma mustelae * * Mycoplasma leonicaptivi Mycoplasma bovirhinis synoviae cluster * Mycoplasma cynos Mycoplasma edwardii Mycoplasma canis Mycoplasma columborale Mycoplasma oxoniensis Mycoplasma citelli Mycoplasma sturni * Mycoplasma pullorum Mycoplasma anatis Mycoplasma crocodyli Mycoplasma alligatoris Mycoplasma hominis homini s group Mycoplasma equirhinis Mycoplasma phocidae * Mycoplasma falconis Mycoplasma spumans Mycoplasma arthritidis * Mycoplasma phocicerebrale * Mycoplasma auris * Mycoplasma alkalescens * Mycoplasma canadense * Mycoplasma gateae hominis cluster Mycoplasma arginini Mycoplasma cloacale Mycoplasma anseris Mycoplasma buccale Mycoplasma hyosynoviae Mycoplasma orale * Mycoplasma indiense Mycoplasma faucium Mycoplasma subdolum Mycoplasma gypis Mycoplasma pulmonis strain UAB CTIP Mycoplasma agassizii pulmonis cluster Mycoplasma testudineum Mycoplasma sualvi Mycoplasma moatsii sualvi cluster Mycoplasma mobile Mycoplasma neurolyticum Mycoplasma cricetuli Mycoplasma collis * Mycoplasma molare Mycoplasma lagogenitalium * Mycoplasma iguanae Mycoplasma hyopneumoniae * Mycoplasma flocculare neurolyticum cluster Mycoplasma ovipneumoniae Mycoplasma dispar Mycoplasma bovoculi Mycoplasma conjunctivae Mycoplasma hyorhinis Mycoplasma vulturis Scale: 0.1 substitutions/site

Figure 109. (Continued) Genus I. Mycoplasma 583

Mycoplasma insons Mycoplasma cavipharyngis fastidiosum cluster Mycoplasma fastidiosum

* hemotropic cluster

Mycoplasma pneumoniae Mycoplasma genitalium Mycoplasma amphoriforme Mycoplasma testudinis pneumoniae cluster Mycoplasma alvi Mycoplasma pirum

pneumoniae group Mycoplasma gallisepticum strain R * Mycoplasma imitans Ureaplasma urealyticum Ureaplasma parvum serovar 3 Ureaplasma gallorale Ureaplasma diversum Ureaplasma cluster Ureaplasma felinum Ureaplasma canigenitalium Mycoplasma penetrans strain HF-2 Mycoplasma iowae Mycoplasma muris muris cluster Mycoplasma microti

Mycoplasma coccoides Mycoplasma haemofelis Mycoplasma haemocanis hemotropic cluster ‘Candidatus Mycoplasma haemobos’ Mycoplasma haemomuris Mycoplasma suis * Mycoplasma wenyonii Mycoplasma ovis

Scale: 0.1 substitutions/site

Figure 109. Phylogenetic relationships in the Mycoplasma hominis and Mycoplasma pneumoniae groups of the order Mycoplasmatales. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Acholeplasma (formerly Mycoplasma) feliminutum was the outgroup. The major groups and clusters are defined in terms of posi- tions in 16S rRNA showing characteristic base composition and signature positions, plus higher-order structural synapomorphies (Johansson and ­Pettersson, 2002; Weisburg et al., 1989). Bootstrap values (100 replicates) <50% are indicated (*); the branching order is considered to be equivocal.

List of species of the genus Mycoplasma

1. Mycoplasma mycoides (Borrel, Dujardin-Beaumetz, Jeantet ca¢pri. L. n. caper, -pri goat; L. gen. n. capri of the goat. AL and Jouan 1910) Freundt 1955, 73 (Asterococcus mycoides Cells are pleomorphic and capable of forming long fila- Borrel, Dujardin-Beaumetz, Jeantet and Jouan 1910, 179) ments and long, helical rods known as rho forms. Nonmo- my.co.i¢des. Gr. n. mukês -êtos mushroom or other fungus; L. tile. An extracellular capsule can be visualized by electron suff. -oides (from Gr. suff. -eides, from Gr. n. eidos that which microscopy following staining with ruthenium red. Colonies is seen, form, shape, figure) resembling, similar; N.L. neut. on solid agar have a characteristic fried-egg appearance and adj. mycoides fungus-like. are notably larger than those of Mycoplasma mycoides subsp. This is the type species of the genus. Cells are pleomor- mycoides. Grows in modified Hayflick medium supplemented phic and capable of forming long filaments. Nonmotile. An with glucose at 37°C. Formation of biofilms has been dem- extracellular capsule can be visualized by electron micros- onstrated (McAuliffe et al., 2006). copy following staining with ruthenium red. Colonies on Pathogenic; causes polyarthritis, mastitis, conjunctivi- solid agar have a characteristic fried-egg appearance. Grows tis (a syndrome collectively termed contagious agalactia), in modified Hayflick medium supplemented with glucose pneumonia, peritonitis, and septicemia in goats; and bal- at 37°C. anitis and vulvitis in sheep. Transmission occurs via direct The species has subsequently been divided as follows. contact between animals or with fomites, or can be vector- borne by the common ear mite (Psoroptes cuniculi). 1a. Mycoplasma mycoides subsp. capri Manso-Silván, Vilei, Tetracyclines are effective therapeutic agents. Eradica- Sachse, Djordjevic, Thiaucourt and Frey 2009, 1357VP (Asterococcus tion from herds is difficult due to the tendency of healthy mycoides var. capri Edward 1953, 874; Mycoplasma mycoides animals to harbor the organism in the ear canal without subsp. mycoides var. large colony Cottew and Yeats 1978, 294) seroconverting. Antigenic cross-reactivity with Mycoplasma 584 Family I. Mycoplasmataceae

Table 137. Descriptive characteristics of species of Mycoplasma a

DNA G+C content Energy Medium pH Representative Species Morphology (mol%) source shift Serum source host Relation to host M. mycoides subsp. mycoides Pleomorphic 24 G A FB Cattle Pathogen M. mycoides subsp. capri Pleomorphic 24 G A FB Goats Pathogen M. adleri Coccoidal 29.6 R K E Goats Pathogen M. agalactiae Coccoidal 29.7 G A FB, E Goats Pathogen M. agassizii Pleomorphic nr G A FB Tortoises Pathogen M. alkalescens Coccobacillary 25.9 R K FB Cattle Pathogen M. alligatoris Coccoidal nr G A FB Alligators Pathogen M. alvi Flask-shaped 26.4 G, R V FB Cattle Commensal M. amphoriforme Flask-shaped 34 G A FB Humans Opportunistic M. anatis Coccoidal 26.6 G A FB Ducks Opportunistic M. anseris Spherical 26 R K E Goose Opportunistic M. arginini Coccoidal 27.6 R K E Mammals Pathogen M. arthritidis Filamentous 30.7 R K FB Rats Pathogen M. auris Pleomorphic 26.9 R K E Goats Commensal M. bovigenitalium Pleomorphic 30.4 OH, OA N FB Cattle Pathogen M. bovirhinis nr 27.3 G A FB Cattle Opportunistic M. bovis Coccobacillary 32.9 OH, OA N FB, E Cattle Pathogen M. bovoculi Coccobacillary 29 G A E Cattle Pathogen M. buccale Coccobacillary 26.4 R K E Humans Commensal M. buteonis Coccoidal 27 G A P Raptors Commensal M. californicum Pleomorphic 31.9 OH, OA N E Cattle Pathogen M. canadense Coccobacillary 29 R K FB Cattle Pathogen M. canis Pleomorphic 28.4 G A FB Dogs Opportunistic M. capricolum subsp. capricolum Coccobacillary 23 G A FB, E Goats Pathogen M. capricolum subsp. Coccobacillary 24.4 G A FB, E Goats Pathogen capripneumoniae M. caviae nr nr G A FB Guinea pigs Commensal M. cavipharyngis Twisted rod 30 G A E Guinea pigs Commensal M. citelli Pleomorphic 27.4 G A FB Squirrels Commensal M. cloacale Spherical 26 R K E Galliforms Commensal M. coccoidesb Coccoidal nr U na na Mice Pathogen M. collis Coccoidal 28 G A E Rodents Commensal M. columbinasale Coccobacillary 32 R K FB Pigeons Commensal M. columbinum Pleomorphic 27.3 R K P Pigeons Commensal M. columborale Coccoidal 29.2 G A P Pigeons Commensal M. conjunctivae Coccobacillary nr G A FB Goats Pathogen M. corogypsi Pleomorphic 28 G A P Vultures Pathogen M. cottewii Coccoid 27 G A E Goats Commensal M. cricetuli Pleomorphic nr G A E Hamsters Commensal M. crocodyli Coccoidal 27.6 G A FB Crocodiles Pathogen M. cynos Coccobacillary 25.8 G A FB Dogs Pathogen M. dispar Pleomorphic 29.3 G A FB, P Cattle Pathogen M. edwardii Coccobacillary 29.2 G A FB Dogs Opportunistic M. elephantis Coccoidal 24 G A E Elephants Commensal M. equigenitalium Pleomorphic 31.5 G A E Horses Opportunistic M. equirhinis Coccobacillary nr R K E Horses Opportunistic M. falconis Coccoidal 27.5 R K P Falcons Opportunistic M. fastidiosum Twisted rod 32.3 G A P Horses Commensal M. faucium Coccoidal nr R K FB Humans Commensal M. felifaucium Coccoidal 31 R K FB, E Pumas Commensal M. feliminutum nr 29.1 G A FB Cats Commensal M. felis Filamentous 25.2 G A FB Cats Pathogen M. fermentans Filamentous 28.7 R, G V FB, E Humans Unclear M. flocculare Coccobacillary 33 U N P Pigs Opportunistic M. gallinaceum Coccobacillary 28 G A P Galliforms Pathogen M. gallinarum Coccobacillary 28 R K P Galliforms Commensal M. gallisepticum Flask-shaped 31 G A FB, E Galliforms Pathogen M. gallopavonis Coccobacillary 27 G A P Turkeys Opportunistic M. gateae nr 28.5 U N FB Cats Opportunistic

(Continued) Genus I. Mycoplasma 585

Table 137. (Continued)

DNA G+C content Energy Medium pH Representative Species Morphology (mol%) source shift Serum source host Relation to host M. genitalium Flask-shaped 31 G A FB Humans Pathogen M. glycophilum Elliptical 27.5 G A E Galliforms Commensal M. gypis Coccoidal 27.1 R K P Vultures Opportunistic M. haemocanisb Coccoidal nr U na na Dogs Pathogen M. haemofelisb Coccoidal 38.8 U na na Cats Pathogen M. haemomurisb Coccoidal nr U na na Mice Opportunistic M. hominis Coccobacillary 33.7 R K FB Humans Pathogen M. hyopharyngis Pleomorphic 24 R K P, E Pigs Commensal M. hyopneumoniae Coccobacillary 28 G A P, FB Pigs Pathogen M. hyorhinis Coccobacillary 27.8 G A FB Pigs Pathogen M. hyosynoviae Pleomorphic 28 G A FB Pigs Pathogen M. iguanae Coccoidal nr G A FB Iguanas Pathogen M. imitans Flask-shaped 31.9 G A FB Ducks, geese Pathogen M. indiense Pleomorphic 32 R K FB Monkeys Commensal M. iners nr 29.6 R K P Galliforms Commensal M. insonsc Twisted rod nr G A FB Iguanas Commensal M. iowae Pleomorphic 25 G, R V FB Turkeys Pathogen M. lagogenitalium Coccoidal 23 G A FB Pikas Commensal M. leachii Pleomorphic nr G A E Cattle Pathogen M. leonicaptivi Pleomorphic 27 G A FB Lions Commensal M. leopharyngis Pleomorphic 28 G A FB Lions Commensal M. lipofaciens Elliptical 24.5 G, R V P Galliforms Commensal M. lipophilum Pleomorphic 29.7 R K FB, E Humans Unclear M. maculosum Coccobacillary 29.6 R K FB, E Dogs Opportunistic M. meleagridis Coccobacillary 28.6 R K P Turkeys Pathogen M. microti Coccoidal nr G A FB Voles Commensal M. moatsii Spheroidal 25.7 G, R V FB, E Monkeys Commensal M. mobile Flask-shaped 25 G, R V E Tench Pathogen M. molare Coccoidal 26 G A FB Dogs Opportunistic M. mucosicanisc Coccoidal nr U nr E Dogs Commensal M. muris Coccoidal 24.9 R K FB Mice Commensal M. mustelae Pleomorphic 28 G A E Minks Commensal M. neurolyticum Filamentous 26.2 G A E Mice Unclear M. opalescens nr 29.2 R K FB Dogs Commensal M. orale Pleomorphic 28.2 R K FB, E Humans Commensal M. ovipneumoniae nr 25.7 G A FB, P Sheep Pathogen M. ovisb Coccoidal nr U na na Sheep Pathogen M. oxoniensis Coccoidal 29 G A FB Hamsters Commensal M. penetrans Flask-shaped 25.7 G, R V FB Humans Opportunistic M. phocicerebrale Dumbbell 25.9 R K FB Seals Pathogen M. phocidae Coccoidal 27.8 G, R V FB, E Seals Opportunistic M. phocirhinis Coccoidal 26.5 R K E, P Seals Pathogen M. pirum Flask-shaped 25.5 G A FB Humans Commensal M. pneumoniae Flask-shaped 40 G A FB Humans Pathogen M. primatum Coccobacillary 28.6 R K FB, E Monkeys Opportunistic M. pullorum Coccobacillary 29 G A P, E Galliforms Pathogen M. pulmonis Flask-shaped 26.6 G A FB, E Mice Pathogen M. putrefaciens Coccobacillary 28.9 G A FB, E Goats Pathogen M. salivarium Coccoidal 27.3 R K E Humans Opportunistic M. simbae Pleomorphic 37 R K FB Lions Commensal M. spermatophilum Coccoidal 32 R K FB Humans Pathogen M. spheniscic Pleomorphic 28 G A P Penguins Pathogen M. spumans Pleomorphic 28.4 R K FB, E Dogs Opportunistic M. sturni Pleomorphic 31 G A FB Songbirds Pathogen M. sualvi Coccobacillary 23.7 R, G V FB Pigs Commensal M. subdolum Coccoidal 28.8 R K FB, E, P Horses Opportunistic M. suisb Coccoidal 31.1 U na na Pigs Pathogen M. synoviae Coccoidal 34.2 G A P Galliforms Pathogen M. testudineum Coccoidal nr G A FB Tortoises Pathogen

(Continued) 586 Family I. Mycoplasmataceae

Table 137. (Continued) DNA G+C content Energy Medium pH Representative Species Morphology (mol%) source shift Serum source host Relation to host M. testudinis Flask-shaped 35 G A FB Tortoises Commensal M. verecundum Pleomorphic 27 OA N FB, E Cattle Commensal “M. vulturis”b, c Coccoidal nr U na na Vultures Unclear M. wenyoniib Coccoidal nr U na na Cattle Pathogen M. yeatsii Coccoidal 26.6 G na FB Goats Opportunistic M. zalophic nr nr G na FB Sea lions Pathogen “Candidatus M. Coccoidal nr U na na Dogs nr haematoparvum”b “Candidatus M. haemobos”b Coccoidal nr U na na Cattle nr “Candidatus M. Coccoidal nr U na na Opossum nr haemodidelphidis”b “Candidatus M. haemolamae”b Coccoidal nr U na na Llamas nr “Candidatus M. Coccoidal nr U na na Cats nr haemominutum”b “Candidatus M. kahaneii”b Coccoidal nr U na na Monkeys nr “Candidatus M. ravipulmonis”b Coccoidal nr U na na Mice Pathogen “Candidatus M. Coccoidal nr U na na Reindeer nr haemotarandirangiferis”b “Candidatus M. turicensis”b Coccoidal nr U na na Cats nr anr, Not reported; na, not applicable; G, glucose; R, arginine; OH, alcohols; OA, organic acids; U, undefined; A, acidic pH shift; K, alkaline pH shift; N, pH remains neutral; V, pH shift can be acidic or alkaline depending on the energy source provided; FB, fetal bovine serum; E, equine serum; P, porcine serum. bNot yet cultivated in cell-free artificial medium. The putative organism “Candidatus M. haemotarandirangiferis” remains to be definitively established and the name has no standing in nomenclature. cHas been grown only in co-culture with eukaryotic cells.

mycoides subsp. mycoides precludes the exclusive reliance Cells are pleomorphic and capable of forming long on serological-based diagnostics. Experimental vaccines filaments, but do not produce rho forms. Nonmotile. An using formalin-inactivated Mycoplasma mycoides subsp. capri extracellular capsule can be visualized by electron micros- appear to protect goats from subsequent challenge (Bar- copy following staining with ruthenium red. Colonies on Moshe et al., 1984; de la Fe et al., 2007). This organism is solid agar have a characteristic fried-egg appearance and under certain quarantine regulations in most non-endemic are notably smaller than those of Mycoplasma mycoides subsp. countries and is a List B pathogen in the World Organiza- capri. Grows in modified Hayflick medium supplemented tion for Animal Health (OIE) disease classification (http:// with glucose at 37°C. Formation of biofilms has been dem- oie.int). onstrated (McAuliffe et al., 2008). Source: isolated from the synovial fluid, synovial mem- Pathogenic; causes a characteristic, highly lethal fibrin- branes, udders, expelled milk, conjunctivae, lungs, blood, ous interstitial pneumonia and pleurisy known as conta- and ear canals of goats; and the urogenital tract of sheep gious bovine pleuropneumonia (CBPP) in adult cattle and (Bergonier et al., 1997; Cottew, 1979; Kidanemariam et al., severe polyarthritis in calves. Transmission occurs primarily 2005; Thiaucourt et al., 1996). via direct contact, but can occur by droplet aerosol as well. Tetracyclines, chloramphenicol, and fluoroquinolones DNA G+C content (mol%): 24 (Tm). Type strain: PG3, NCTC 10137, CIP 71.25. are effective chemotherapeutic agents; however, treat- Sequence accession nos (16S rRNA gene): U26037 (strain ment of endemic herds is often counterproductive as resis- PG3T), U26044 (strain Y-goat). tance can develop in carrier animals. Organisms are often Further comment: Mycoplasma mycoides subsp. capri now sequestered in areas of coagulative necrosis in subclinically refers to strains once known as Mycoplasma mycoides subsp. infected animals and can serve as a reservoir for reintroduc- mycoides var. large colony as well as strains known as Myco- tion of resistant clones of Mycoplasma mycoides subsp. mycoides plasma mycoides subsp. capri (Manso-Silván et al., 2009; into a herd. Culling of infecting herds and restricting the ­Shahram et al., 2010). movement of infected animals are more effective strategies for controlling spread of the disease (Windsor and Masiga, 1b. Mycoplasma mycoides subsp. mycoides Manso-Silván, Vilei, 1977). Killed and live vaccines are available for the preven- Sachse, Djordjevic, Thiaucourt and Frey 2009, 1356VP (Myco- tion of infection, but suffer from low antigenicity, poor plasma mycoides subsp. mycoides var. small colony Cottew and efficacy, and residual pathogenesis (Brown et al., 2005). Yeats 1978, 294) Antigenic cross-reactivity with Mycoplasma mycoides subsp. my.co.i¢des. Gr. n. mukês -êtos mushroom or other fungus; L. capri and Mycoplasma leachii preclude the exclusive reliance suff. -oides (from Gr. suff. -eides from Gr. n. eidos that which on serological-based diagnostics. Multiple molecular diag- is seen, form, shape, figure) resembling, similar; N.L. neut. nostics have been described (Gorton et al., 2005; Loren- adj. mycoides fungus-like. zon et al., 2008; Persson et al., 1999) and many ­additional Genus I. Mycoplasma 587

molecular tools such as insertion sequence ­typing have led strains and the tendency of antimicrobials to be excreted to a greater understanding of the epidemiology of outbreaks in milk. Control measures such as disinfection of fomites (Cheng et al., 1995; Frey et al., 1995; Vilei et al., 1999). This (endemic areas) and culling of infected animals (acute out- organism is under certain quarantine regulations in most breaks) are more common practices. Mycoplasma agalactiae non-endemic countries and is listed in the Terrestrial Ani- reportedly shares surface antigens with Mycoplasma bovis mal Health Code of the Office International des Epizooties and Mycoplasma capricolum subsp. capricolum (Alberti et al., (http://oie.int). 2008; Boothby et al., 1981), potentially complicating serol- Source: isolated from the lungs, pleural fluid, lymph ogy-based diagnosis of infection. Molecular diagnostics that nodes, sinuses, kidneys, urine, synovial fluid, and syn- can distinguish Mycoplasma agalactiae from Mycoplasma bovis ovial membranes of cattle and water buffalo (Gourlay and have been described (Chávez Gonzalez et al., 1995). Com- ­Howard, 1979; Scanziani et al., 1997; Scudamore, 1976); mercially available vaccines are widely used, but exhibit the respiratory tract of bison; the respiratory tract of yak; poor efficacy. Numerous experimental vaccines have been and the lungs, nasopharynx, and pleural fluid of sheep and described. This organism is under certain quarantine regu- goats (Brandao, 1995; Kusiluka et al., 2000). lations in some countries and is listed in the Terrestrial Ani- T DNA G+C content (mol%): 26.1 (Tm), 24.0 (strain PG1 mal Health Code of the Office International des Epizooties complete genome sequence). (http://oie.int). Type strain: PG1, NCTC 10114, CCUG 32753. Source: isolated from the joints, udders, milk, conjuncti- Sequence accession nos: U26039 (16S rRNA gene), vae, lungs, vagina, liver, spleen, kidneys, and small intestine BX293980 (strain PG1T complete genome sequence). of sheep and goats. T Further comment: Mycoplasma mycoides subsp. mycoides now DNA G+C content (mol%): 30.5 (Tm), 29.7 (strain PG2 refers exclusively to the agent of CBPP (Manso-Silván et al., complete genome sequence). 2009). Type strain: PG2, NCTC 10123, CIP 59.7. Sequence accession nos: M24290 (16S rRNA gene), 2. Mycoplasma adleri Del Giudice, Rose and Tully 1995, 31VP NC_009497 (strain PG2T complete genome sequence). ad¢le.ri. N.L. masc. gen. n. adleri of Adler, referring to Henry Adler, a Californian veterinarian whose studies contributed 4. Mycoplasma agassizii Brown, Brown, Klein, McLaughlin, VP much new information concerning the pathogenic role of Schumacher, Jacobson, Adams and Tully 2001c, 417 caprine and avian mycoplasmas. a.gas.si¢zi.i. N.L. masc. gen. n. agassizii of Agassiz, referring Cells are primarily coccoid. Nonmotile. Colonies on solid to Louis Agassiz, a naturalist whose name was assigned to a media have a typical fried-egg appearance. Grows well in species of desert tortoise (Gopherus agassizii) from which the Hayflick medium supplemented with arginine at 35–37°C. organism was isolated. Pathogenic; associated with suppurative arthritis and Cells are coccoid to pleomorphic, with some strains joint abscesses. Mode of transmission is unknown. appearing to possess a rudimentary terminal structure. Cells Source: isolated from an abscessed joint of a goat with sup- exhibit gliding motility. Colony forms on solid medium vary purative arthritis (Del Giudice et al., 1995). from those with a fried-egg appearance to some with mul- DNA G+C content (mol%): 29.6 (Bd). berry characteristics. Grows well in SP-4 medium supple- Type strain: G145, ATCC 27948, CIP 105676. mented with glucose at 30°C. Sequence accession no. (16S rRNA gene): U67943. Pathogenic; causes chronic upper respiratory tract dis- ease characterized by severe rhinitis in desert tortoises, 3. Mycoplasma agalactiae (Wróblewski 1931) Freundt 1955, gopher tortoises, Russian tortoises, and leopard tortoises. 73AL (Anulomyces agalaxiae Wróblewski 1931, 111) Mode of transmission appears to be intranasal inhalation a.ga.lac.ti¢ae. Gr. n. agalactia want of milk, agalactia; N.L. (Brown et al., 1994). gen. n. agalactiae of agalactia. Source: isolated from the nares and choanae of desert Cells are primarily coccoid, but are occasionally branched tortoises, gopher tortoises, Russian tortoises, and leopard and filamentous. Nonmotile. Colonies on solid media have tortoises (Brown et al., 2001c). a typical fried-egg appearance. Grows well in SP-4 or Hay- DNA G+C content (mol%): not determined. flick medium supplemented with glucose at 37°C. Forma- Type strain: PS6, ATCC 700616. tion of biofilms has been demonstrated (McAuliffe et al., Sequence accession no. (16S rRNA gene): U09786. 2006). 5. Mycoplasma alkalescens Leach 1973, 149AL Pathogenic; causes polyarthritis, mastitis, conjunctivi- tis (a syndrome collectively termed contagious agalactia; al.ka.les¢cens. N.L. v. alkalesco to make alkaline, referring to Bergonier et al., 1997), nonsuppurative arthritis, pneumo- the reaction produced in arginine-containing media; N.L. nia, abortion, and granular vulvovaginitis (Cottew, 1983; part. adj. alkalescens alkaline-making. DaMassa, 1996) in goats and sheep. Transmission occurs via Coccoid to coccobacillary cells. Motility and colony mor- direct contact, most commonly during feeding (kids and phology have not been described for this species. Grows lambs) or milking (dams and ewes). well in SP-4 medium supplemented with arginine at 37°C. Macrolides and fluoroquinolones are effective chemo- Pathogenic; causes febrile arthritis and sometimes masti- therapeutic agents; however, antimicrobial therapy is not tis, pneumonia, and otitis in cattle (Kokotovic et al., 2007; often utilized in widespread outbreaks due to the potential Lamm et al., 2004; Leach, 1973). Mode of transmission has for infected animals to develop carrier states with resistant not been established. 588 Family I. Mycoplasmataceae

Tetracyclines and pleuromutilins are effective chemother- Cells are primarily coccoid; however, subsets of the apeutic agents (Hirose et al., 2003). Mycoplasma alkalescens ­population display elongated, flask-shaped cells with well- reportedly shares surface antigens with many arginine- defined terminal structures. Unlike most species that exhibit ­fermenting Mycoplasma species; however, this is only likely polar structures, Mycoplasma alvi appears to be nonmotile to interfere with accurate diagnosis of infection in the case (Bredt, 1979; Hatchel and Balish, 2008). Colonies on solid of Mycoplasma arginini. medium exhibit typical fried-egg morphology. Grows well in Source: isolated from the synovial fluid, expelled milk, SP-4 medium supplemented with either glucose or arginine lungs, ears, prepuce, and semen of cattle. at 37°C.

DNA G+C content (mol%): 25.9 (Tm). No evidence of pathogenicity. Mode of transmission has Type strain: D12, PG51, NCTC 10135, ATCC 29103. not been established definitively. Sequence accession no. (16S rRNA gene): U44764. Source: isolated from the lower alimentary tract, feces, Further comment: Bovine serogroup 8 of Leach (1967). bladder, and vagina of cows, and the intestinal tract of voles (Gourlay and Howard, 1979). 6. Mycoplasma alligatoris Brown, Farley, Zacher, Carlton, Clip- DNA G+C content (mol%): 26.4 (Bd). pinger, Tully and Brown 2001a, 423VP Type strain: Ilsley, NCTC 10157, ATCC 29626. al.li.ga.to¢ris. N.L. n. alligator, -oris an alligator; N.L. gen. n. Sequence accession no. (16S rRNA gene): U44765. alligatoris of/from an alligator. 8. Mycoplasma amphoriforme Pitcher, Windsor, Windsor, Cells are primarily coccoid. Nonmotile. Colonies on Bradbury, Yavari, Jensen, Ling and Webster 2005, 2592VP solid medium exhibit typical fried-egg morphology. Growth am.pho.ri.for¢me. L. n. amphora amphora; L. adj. suff. is very rapid in SP-4 medium supplemented with glucose -formis -e like, of the shape of; N.L. neut. adj. amphoriforme at 30°C. amphora-shaped, having the form of an amphora. Pathogenic; causes a multisystemic inflammatory illness with a lethality unprecedented among mycoplasmas. Patho- Cells are flask-shaped with a distinct terminal structure logic lesions in infected animals include meningitis, intersti- reminiscent of Mycoplasma gallisepticum. Cells exhibit low- tial pneumonia, fibrinous pleuritis, polyserositis, fibrinous speed gliding motility and move in the direction of the ter- pericarditis, myocarditis, endocarditis, synovitis, and splenic minal structure (Hatchel et al., 2006). Colony morphology and hepatic necrosis. A high level of mortality of naturally variable, from a typical fried-egg morphology to a “ground and experimentally infected American alligators (Alligator glass” appearance. Grows well in SP-4 medium supple- missisippiensis) and broad-nosed caimans (Caiman latiro- mented with glucose at 37°C. stris) occurs. In contrast, Mycoplasma alligatoris colonizes Pathogenicity and mode of transmission have not been the tonsils of experimentally infected Siamese crocodiles established definitively. (Crocodylus siamensis) without causing overt pathology. Such Despite showing sensitivity to fluoroquinolones, tetra- findings led to the hypothesis that Mycoplasma alligatoris is cyclines, and macrolides in vitro, Mycoplasma amphoriforme a natural commensal of a species more closely related to appears to be successfully evasive during treatment of crocodiles than alligators and that the extreme disease state patients with these antibiotics. The veterinary antibiotic observed in alligators results from an enzoonotic infection valnemulin is successful at controlling infection (Webster (Brown et al., 1996; Pye et al., 2001). The natural mode of et al., 2003). transmission has not been established definitively; however, Source: isolated from the sputum of immunocompro- animals can be experimentally infected via inoculation of mised humans with bronchitis and related lower respira- the glottis. tory tract disease (Pitcher et al., 2005; Webster et al., 2003). Source: isolated from the blood, synovial fluid, cerebrospi- The prevalence of Mycoplasma amphoriforme in the general nal fluid, lungs, brain, heart, liver, and spleen of naturally human population is unsubstantiated. and experimentally infected American alligators, experi- DNA G+C content (mol%): 34.0 (fluorescent intensity). mentally infected broad-nosed caimans, and from the ton- Type strain: A39, NCTC 11740, ATCC BAA-992. sils of experimentally infected Siamese crocodiles. Sequence accession no. (16S rRNA gene): FJ226575. DNA G+C content (mol%): not determined. 9. Mycoplasma anatis Roberts 1964, 471AL Type strain: A21JP2, ATCC 700619. a.na¢tis. L. n. anas, -atis a duck; L. gen. n. anatis of a duck. Sequence accession no. (16S rRNA gene): U56733. Further comment: the name Mycoplasma alligatoris was Cells have been described as coccoid with ring forms, but assigned for this organism in consideration of the initial cellular and colony morphology is generally poorly described. isolation from an alligator (Order: Crocodylia). The Eng- Motility for this species has not been assessed. Grows well in lish word “alligator” is from the Spanish el lagarto (Latin SP-4 medium supplemented with glucose 37°C. ille lacertu the lizard). However, the specific epithet lacerti, Isolated from pathologic lesions, but attempts to repro- originally proposed for this taxon, was ultimately rejected duce disease following experimental infection have been because of the modern phylogenetic distinction between equivocal (Amin and Jordan, 1978; Roberts, 1964). The lizards (Order: Lacertilia) and crocodilians. mode of transmission has not been established definitively. Source: isolated from pathologic lesions, the respiratory 7. Mycoplasma alvi Gourlay, Wyld and Leach 1977, 95AL tract, hock joint, pericardium, cloaca, and meninges of al¢vi. L. n. alvus bowel, womb, stomach; L. gen. n. alvi of ducks (Goldberg et al., 1995; Ivanics et al., 1988; Tiong, the bowel. 1990). Genus I. Mycoplasma 589

DNA G+C content (mol%): 26.6 (Bd). have a typical fried-egg appearance. Grows well in SP-4 Type strain: 1340, ATCC 25524, NCTC 10156. medium supplemented with arginine. Sequence accession no. (16S rRNA gene): AF412970. Pathogenic; causes purulent polyarthritis, rhinitis, ­otitis media, ocular lesions, and abscesses in rats. Mycoplasma 10. Mycoplasma anseris Bradbury, Jordan, Shimizu, Stipkovits arthritidis is also known to superinfect lung lesions initiated and Varga 1988, 76VP by Mycoplasma pulmonis. Experimental inoculations via vari- an¢se.ris. L. gen. n. anseris of the goose. ous routes can result in septicemia, acute flaccid paralysis, Cells are primarily spherical. Nonmotile. Colonies on and pyelonephritis in rats, and chronic arthritis in mice and solid medium have a typical fried-egg appearance. Grows rabbits. Mycoplasma arthritidis is unique among mycoplasmas well in Hayflick medium supplemented with arginine at in harboring the lysogenized bacteriophage MAV1, whose 37°C. contribution to virulence is equivocal, and producing the Opportunistic pathogen; associated with balanitis of potent mitogen MAM, which appears to confer increased geese, but can be isolated from clinically normal animals. toxicity and lethality but to be irrelevant to arthritogenicity Mode of transmission has not been established definitively. (Clapper et al., 2004; Luo et al., 2008; Voelker et al., 1995). Source: isolated from the phallus and cloaca of geese The mechanism of transmission is largely dependent on the (Hinz et al., 1994; Stipkovits et al., 1984a). tissue infected.

DNA G+C content (mol%): 24.7–26.0 (Bd/Tm). Source: isolated from the synovial membranes, synovial Type strain: 1219, ATCC 49234. fluid, middle ear, eye, abscessed bone, abscessed ovary, and Sequence accession no. (16S rRNA gene): AF125584. oropharynx of wild and captive rats. Isolations have also been reported from non-human primates including rhesus 11. Mycoplasma arginini Barile, Del Giudice, Carski, Gibbs and monkeys and bush babies (Somerson and Cole, 1979); and Morris 1968, 490AL from joint fluid of wild boars (Binder et al., 1990). The true ar.gi.ni¢ni. N.L. n. argininum arginine, an amino acid; N.L. origins of putative isolates from the human urethra, pros- gen. n. arginini of arginine, referring to its hydrolysis. tate, and cervix have been questioned (Cassell and Hill, Cells are primarily coccoid. Motility for this species has 1979; Washburn et al., 1995). T not been assessed. Colonies have either the typical fried-egg DNA G+C content (mol%): 30.0 (Tm; strain PG6 ), 30.7 morphology or a granular, “berry-like” appearance. Grows (strain 158L3-1 complete genome sequence). well in modified Hayflick medium supplemented with argi- Type strain: PG6, ATCC 19611, NCTC 10162, CIP 104678, nine at 37°C. NBRC 14860. Pathogenic; associated with pneumonia, vesiculitis, kera- Sequence accession nos: M24580 (16S rRNA gene), toconjunctivitis, and mastitis in cattle; pneumonia and ker- CP001047 (strain 158L3-1 complete genome sequence). atoconjunctivitis in sheep; possibly with arthritis in goats; 13. Mycoplasma auris DaMassa, Tully, Rose, Pitcher, Leach and and septicemia in immunocompromised humans (Tully Cottew 1994, 483VP and Whitcomb, 1979; Yechouron et al., 1992). Modes of transmission have not been definitively assessed and likely au¢ris. L. gen. n. auris of the ear, referring to the provenance vary by anatomical site. of the organism, the ears of goats. Mycoplasma arginini is commonly associated with contam- Cells are coccoid to pleomorphic. Nonmotile. Colo- ination of eukaryotic cell culture and is frequently removed nies on solid medium have a typical fried-egg appearance. by treatment of cells with antibiotics and/or maintenance of Grows well in Hayflick medium supplemented with argin- cell lines in antibiotic-containing medium. The most effec- ine at 37°C. tive classes of antibiotics for cell culture eradication are tet- No evidence of pathogenicity. Mechanism of transmis- racyclines, macrolides, and fluoroquinolones. Additionally, sion has not been established. passage of eukaryotic cells in hyperimmune serum raised Source: isolated from the external ear canals of goats against Mycoplasma arginini has been shown to be an effec- (Damassa et al., 1994).

tive method of eradication (Jeansson and Brorson, 1985). DNA G+C content (mol%): 26.9 (Tm). Source: isolated from a wide array of mammalian hosts Type strain: UIA, ATCC 51348, NCTC 11731, CIP including cattle, sheep, goats, pigs, horses, domestic dogs, 105677. domestic cats, lions, lynxes, cheetahs, chamois, camels, Sequence accession no. (16S rRNA gene): U67944. ibexes, humans, and mice. 14. Mycoplasma bovigenitalium Freundt 1955, 73AL DNA G+C content (mol%): 27.6 (Tm). Type strain: G230, ATCC 23838, NCTC 10129, CIP 71.23, bo.vi.ge.ni.ta¢li.um. L. n. bos, bovis the ox, bull, cow; L. pl. NBRC 14476. n. genitalia the genitals; N.L. pl. gen. n. bovigenitalium of Sequence accession no. (16S rRNA gene): U15794. bovine genitalia. Cells range from coccoid to filamentous. Motility for this 12. Mycoplasma arthritidis (Sabin 1941) Freundt 1955, 73AL species has not been assessed. Colonies on solid medium (Murimyces arthritidis Sabin 1941, 57) have a typical fried-egg appearance. Grows well in SP-4 ar.thri¢ti.dis. Gr. n. arthritis -idos gout, arthritis; N.L. gen. n. broth supplemented with glucose and/or arginine at 37°C arthritidis of arthritis. and produces a “film and spots” reaction. Cells are filamentous and vary in length. Motility for this Pathogenic; causes vulvovaginitis, vesiculitis, epididymi- species has not been assessed. Colonies on solid medium tis, abortion, infertility, mastitis, pneumonia, conjunctivitis, 590 Family I. Mycoplasmataceae

and arthritis in cattle; and pneumonia and conjunctivitis in mastitis, and arthritis in goats (Egwu et al., 2001; Gourlay domestic dogs. Mode of transmission is via sexual contact and Howard, 1979). Mode of transmission is via direct con- and/or droplet aerosol. tact with infected animals or fomites, most commonly dur- Control measures during outbreaks of Mycoplasma bovi- ing feeding (suckling or trough), milking (cows), aerosol, genitalium infection include suspension of natural breeding or sexual contact. in favor of artificial insemination with disposable instru- Macrolides and fluoroquinolones are effective chemo- ments and, in severe cases, culling of infected animals. therapeutic agents in vitro; however, antimicrobial therapy Source: isolated from the udders, seminal vesicles, pre- is not often utilized in animals with advanced disease due puce, semen, vagina, cervix, lungs, conjunctivae, and joint to poor efficacy and the tendency of antimicrobials to be capsule of cattle; and from the lungs, prepuce, prostate, excreted in milk. Control measures such as disinfection vagina, cervix, and conjunctivae of domestic dogs (Chalker, of fomites, isolation of infected animals, and euthanasia 2005; Gourlay and Howard, 1979). of animals showing clinical signs are more common prac-

DNA G+C content (mol%): 30.4 (Tm). tices. Mycoplasma bovis reportedly shares surface antigens Type strain: PG11, ATCC 19852, NCTC 10122, NBRC with Mycoplasma agalactiae (Boothby et al., 1981), poten- 14862. tially complicating serology-based diagnosis of infection. Sequence accession nos (16S rRNA gene): M24291, Molecular diagnostics that can distinguish Mycoplasma aga- AY121098. lactiae from Mycoplasma bovis have been described (Chávez Further comment: the collection of strains formerly referred ­Gonzalez et al., 1995). Several vaccines are commercially to as “Mycoplasma ovine/caprine serogroup 11” have been available, but exhibit poor efficacy in that they tend to allow reclassified as Mycoplasma bovigenitalium (Nicholas et al., for the establishment of infection while only preventing 2008). overt clinical signs. Source: isolated from the udders, expelled milk, synovial 15. Mycoplasma bovirhinis Leach 1967, 313AL fluid, synovial membranes, conjunctivae, lungs, ear canals, bo.vi.rhi¢nis. L. n. bos, bovis the ox; Gr. n. rhis, rhinos nose; tympanic membranes, aborted calves, uterus, cervix, vagina, N.L. gen. n. bovirhinis of the nose of the ox. and semen of cattle; from the lungs and synovial fluid of Cell and colony morphology and motility for this spe- bison; and from the lungs and udders of goats.

cies are poorly defined. Grows well in SP-4 medium supple- DNA G+C content (mol%): 32.9 (Tm). mented with glucose at 37°C. Type strain: Donetta, PG45, ATCC 25523, NCTC 10131. Pathogenic; causes pneumonia, otitis, conjunctivitis, and Sequence accession no. (16S rRNA gene): AJ419905. mastitis in cattle. Mycoplasma bovirhinis is often found in co- Further comment: Bovine serotype 5 of Leach (1967). infections with other pathogens, leading to speculation that AL it often acts as a superinfecting agent. Mode of transmission 17. Mycoplasma bovoculi Langford and Leach 1973, 1443 has not been established definitively. bo.vo¢cu.li. L. n. bos, bovis ox, bull, cow; L. n. oculus the eye; Source: isolated from the lungs, nasopharynx, trachea, N.L. gen. n. bovoculi of the bovine eye. udders, expelled milk, ears, conjunctivae, and rarely from Cells are coccoid to coccobacillary. Motility for this the urogenital tract of cattle (Gourlay and Howard, 1979). species has not been assessed. Colonies on solid medium DNA G+C content (mol%): 27.3 (Tm). have a typical fried-egg appearance. Grows well in Hayflick Type strain: PG43, ATCC 27748, NCTC 10118, CIP 71.24, medium supplemented with glucose at 37°C. NBRC 14857. Pathogenic; causes conjunctivitis and keratoconjunctivi- Sequence accession no. (16S rRNA gene): U44766. tis in cattle. Face flies are the suggested mechanism of trans- 16. Mycoplasma bovis (Hale, Hemboldt, Plastridge and Stula mission, though this has yet to be established definitively. 1962) Askaa and Ernø 1976, 325AL (Mycoplasma agalactiae Topical application of oxytetracycline is an effective treat- var. bovis Hale, Hemboldt, Plastridge and Stula 1962, 591; ment for infection. Mycoplasma bovimastitidis Jain, Jasper and Dellinger 1967, Source: isolated from the conjunctivae and semen of cat- 409) tle, and from aborted calves (Langford and Leach, 1973; Singh et al., 2004). bo¢vis. L. n. bos the ox; L. gen. n. bovis of the ox. DNA G+C content (mol%): 29.0 (Tm). Cells range from coccoidal to short filaments. Nonmotile. Type strain: M165/69, NCTC 10141, ATCC 29104. Formation of biofilms has been demonstrated (­McAuliffe Sequence accession no. (16S rRNA gene): U44768. et al., 2006). Colonies on solid medium have the typical Further comment: Mycoplasma bovoculi was originally fried-egg appearance, with notably large centers. Grows described as Mycoplasma oculi by Leach in 1973, wherein well in SP-4 medium supplemented with glucose at 37°C the defining publication referring to the species as Myco- and produces a “film and spots” reaction. plasma bovoculi (Langford and Leach, 1973) was cited as “in Pathogenic; causes mastitis, polyarthritis, keratoconjunc- press”. tivitis (Gourlay and Howard, 1979), pneumonia, and otitis media (Caswell and Archambault, 2007; Maeda et al., 2003), 18. Mycoplasma buccale Freundt, Taylor-Robinson, Purcell, and is rarely associated with infertility, abortion, endometri- Chanock and Black 1974, 252AL tis, salpingitis, and vesiculitis (Doig, 1981; Gourlay and How- buc.ca¢le. L. n. bucca the mouth; L. neut. suff. -ale suffix ard, 1979) in cattle; pneumonia and polyarthritis in bison denoting pertaining to; N.L. neut. adj. buccale buccal, per- (Dyer et al., 2008); and is rarely associated with pneumonia, taining to the mouth. Genus I. Mycoplasma 591

Cells range from coccoid to filamentous. Motility for this Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid medium ­species has not been assessed. Colonies on solid agar have have a typical fried-egg appearance. Grows well in Hayflick a characteristic fried-egg appearance. Grows well in SP-4 medium supplemented with arginine and herring sperm medium supplemented with arginine at 37°C. DNA at 37°C. Pathogenic; causes mastitis and arthritis, and may be asso- No evidence of pathogenicity. Mode of transmission has ciated with infertility, abortion, and pneumonia of cattle. not been assessed definitively. Mode of transmission has not been established definitively. Source: isolated from the oropharynx of humans, rhesus Source: isolated from the udders, expelled milk, synovial macaques, chimpanzees, orangutans, baboons, African membranes, aborted calves, vagina, semen, and lungs green monkeys, crab-eating macaques, and patas monkeys (Boughton et al., 1983; Friis and Blom, 1983; Gourlay and (Somerson and Cole, 1979). Howard, 1979; Jackson et al., 1981).

DNA G+C content (mol%): 26.4 (Tm). DNA G+C content (mol%): 29.0 (Tm). Type strain: CH20247, ATCC 23636, NCTC 10136, CIP Type strain: 275C, NCTC 10152, ATCC 29418. 105530, NBRC 14851. Sequence accession no. (16S rRNA gene): U44769. Sequence accession no. (16S rRNA gene): AF125586. 22. Mycoplasma canis Edward 1955, 90AL 19. Mycoplasma buteonis Poveda, Giebel, Flossdorf, Meier and ca¢nis. L. n. canis, -is a dog; L. gen. n. canis of a dog. Kirchhoff 1994, 97VP Cells are pleomorphic, exhibiting branched and filamen- bu.te.o¢nis. L. masc. n. buteo, -onis buzzard; L. masc. gen. n. tous forms. Motility for this species has not been assessed. buteonis of the buzzard. Colonies on solid medium exhibit two stable forms: “smooth” Cells are coccoid. Motility for this species has not been colonies with a nongranular appearance and round edges; assessed. Colonies on solid medium have typical fried-egg and “rough” colonies with a granular appearance and irreg- appearance. Grows well in modified Frey’s medium supple- ular or crenated edges. Each form maintains its characteris- mented with glucose at 37°C. tic appearance during repeated subculturing. Grows well in Mycoplasma buteonis may be pathogenic for saker falcons, SP-4 medium supplemented with glucose at 37°C. as it was found in the respiratory tract, nervous system, and Opportunistic pathogen; associated with infertility and bone of a nestling with pneumonia, hepatitis, ataxia, and adverse pregnancy outcomes, endometritis, epididymi- dyschondroplasia. No evidence of pathogenicity for buz- tis, urethritis, cystitis, and pneumonia of domestic dogs zards. Mode of transmission has not been assessed. (Chalker, 2005); pneumonia of cattle (ter Laak et al., Source: isolated from the trachea of buzzards; from the 1992b); and pneumonia in immunocompromised humans eggs of the lesser kestrel; and the trachea, lungs, brain, and (Armstrong et al., 1971). Mode of transmission is via sex- bone marrow of the saker falcon. Has been detected in the ual contact or aerosol. Mycoplasma canis appears to have a common kestrel and the Western marsh harrier (Erdélyi greater tendency toward upper respiratory tract commen- et al., 1999; Lierz et al., 2008a, 2008c). salism and urogenital tract pathogenicity in dogs, while DNA G+C content (mol%): 27.0 (Bd). exhibiting pathogenicity for the respiratory tract of cattle. Type strain: Bb/T2g, ATCC 51371. Source: isolated from the cervix, vagina, prepuce, Sequence accession no. (16S rRNA gene): AF412971. epididymis, prostate, semen, urine, bladder, oropharynx, nares, lungs, trachea, conjunctivae, kidneys, spleen, peri- 20. Mycoplasma californicum Jasper, Ernø, Dellinger and cardium, liver, and lymph nodes of domestic dogs; from the VP Christiansen 1981, 344 lungs and oropharynx of cattle; from the lungs and phar- ca.li.for¢ni.cum. N.L. neut. adj. californicum pertaining to ynx of humans; and from the throat and rectum of baboons California. and African green monkeys.

Cells are coccoid to filamentous. Motility for this spe- DNA G+C content (mol%): 28.4 (Tm). cies has not been assessed. Colonies are conical in shape Type strain: PG14, ATCC 19525, NCTC 10146, NBRC with distinct small centers. Grows well in modified Hayflick 14846. broth at 37°C. Sequence accession no. (16S rRNA gene): AF412972. Pathogenic; causes purulent mastitis in cows and rarely 23. Mycoplasma capricolum Tully, Barile, Edward, Theodore in sheep. Mode of transmission has not been established and Ernø 1974, 116AL definitively. ca.pri.co¢lum. L. n. caper, -pri the male goat; N.L. -suff. colus, Source: isolated from the udders and expelled milk of -a, -um (from L. v. incolere to dwell) dwelling; N.L. neut. adj. cows and ewes. capricolum dwelling in a male goat. DNA G+C content (mol%): 31.9 (Bd). Type strain: ST-6, ATCC 33461, AMRC-C 1077, NCTC Cells are coccobacillary. Nonmotile. Colonies on solid 10189. agar have a characteristic fried-egg appearance. Grows in Sequence accession no. (16S rRNA gene): M24582. SP-4 or modified Hayflick medium supplemented with glu- cose at 37°C. 21. Mycoplasma canadense Langford, Ruhnke and Onoviran DNA G+C content (mol%): 24.1 (T ). AL m 1976, 218 Type strain: California kid, ATCC 27343, NCTC 10154, ca.na.den¢se. N.L. neut. adj. canadense pertaining to CIP 104620. ­Canada. The species has subsequently been divided as follows. 592 Family I. Mycoplasmataceae

23a. Mycoplasma capricolum subsp. capricolum (Tully, Barile, undertaken. Culling of infected herds and restricting the Edward, Theodore and Ernø 1974) Leach, Ernø and movement of infected animals are more common strate- MacOwan 1993, 604VP (Mycoplasma capricolum Tully, Barile, gies for controlling spread of the disease (Thiaucourt et al., Edward, Theodore and Ernø 1974, 116) 1996). Live and killed vaccines have been described; how- ca.pri.co¢lum. L. n. caper, -pri the male goat; N.L. -suff. colus, ever, each appear to afford delayed or partial protection -a, -um (from L. v. incolere to dwell) dwelling; N.L. neut. adj. from morbidity, and few have been completely successful capricolum dwelling in a male goat. at preventing infection (Browning et al., 2005). Antigenic cross-reactivity with Mycoplasma capricolum subsp. capri- Cells are coccobacillary and can produce long, helical colum and Mycoplasma leachii preclude the exclusive reli- rods known as rho forms. Nonmotile. Colonies on solid ance on serological-based diagnostics. Multiple molecular agar have a characteristic fried-egg appearance. Grows in diagnostics have been described (Lorenzon et al., 2008; SP-4 or modified Hayflick medium supplemented with glu- March et al., 2000; Woubit et al., 2004). This organism is cose at 37°C. under certain quarantine regulations in some countries Pathogenic; causes fibrinopurulent polyarthritis, mastitis, and is listed in the Terrestrial Animal Health Code of the conjunctivitis (a syndrome collectively termed contagious Office International des Epizooties (http://www.oie.int). agalactia) and septicemia in goats. Transmission occurs via Source: isolated from the lower respiratory tract of goats, direct contact between animals or with fomites. sheep, mouflon, and ibex. Tetracyclines, macrolides, and tylosin are effective che- DNA G+C content (mol%): 24.4 (Bd). motherapeutic agents. Treatment of acutely infected Type strain: F38, NCTC 10192. animals often leads to the eradication of the organism, Sequence accession no. (16S rRNA gene): U26042. whereas treatment of chronically infected animals does not. Further comment: previously known as the F38-type caprine Early intervention with antibiotics and improved sanitation mycoplasmas. are effective control measures (Thiaucourt et al., 1996). Antigenic cross-reactivity with Mycoplasma capricolum subsp. 24. Mycoplasma caviae Hill 1971, 112AL capripneumoniae and Mycoplasma leachii preclude the exclu- ca.vi¢ae. N.L. n. cavia guinea pig (Cavia cobaya); N.L. gen. n. sive reliance on serological-based diagnostics. Multiple caviae of a guinea pig. molecular diagnostics have been described (Fitzmaurice Cell morphology for this species has not been described et al., 2008; Greco et al., 2001). This organism is under cer- and motility has not been assessed. Colonies on solid agar tain quarantine regulations in some countries and is listed have a characteristic fried-egg appearance. Grows in SP-4 in the Terrestrial Animal Health Code of the Office Interna- medium supplemented with glucose at 37°C. tional des Epizooties (http://oie.int). No evidence of pathogenicity. Mode of transmission has Source: isolated from the synovial fluid, synovial mem- not been assessed definitively. branes, udders, expelled milk, conjunctivae, spleen, Source: isolated from the nasopharynx and urogenital nasopharynx, oral cavity, and ear canal of goats; and the tract of guinea pigs (Hill, 1971). nasopharynx of sheep (Cottew, 1979). DNA G+C content (mol%): not determined. DNA G+C content (mol%): 24.1 (T ), 23 (strain California m Type strain: G122, ATCC 27108, NCTC 10126. kidT complete genome). Sequence accession no. (16S rRNA gene): AF221111. Type strain: California kid, ATCC 27343, NCTC 10154, CIP 104620. 25. Mycoplasma cavipharyngis Hill 1989, 371VP (Effective publi- Sequence accession nos: U26046 (16S rRNA gene), cation: Hill 1984, 3187) NC_007633 (strain California kidT complete genome). ca.vi.pha.ryn¢gis. N.L. n. cavia the guinea pig (Cavia cobaya); 23b. Mycoplasma capricolum subsp. capripneumoniae Leach, N.L. n. pharynx -yngis (from Gr. n. pharugx pharuggos throat) Ernø and MacOwan 1993, 604VP throat; N.L. gen. n. cavipharyngis of the throat of a guinea pig. ca.pri.pneu.mo.ni¢ae. L. n. capra, -ae a goat; Gr. n. pneumonia disease of the lungs, pneumonia; N.L. gen. n. capripneumo- Cells are highly filamentous and filaments are twisted at niae of a pneumonia of a goat. intervals along their length. Regular helical forms like those of Spiroplasma species are not produced. Nonmotile. Growth Cells are coccobacillary. Nonmotile. Colonies on solid on solid medium shows small, granular colonies with poorly agar have a characteristic fried-egg appearance. Grows in defined centers. Grows in Hayflick medium supplemented SP-4 or modified Hayflick medium supplemented with glu- with glucose at 35°C. cose at 37°C. No evidence of pathogenicity. Mode of transmission has Pathogenic; causes characteristic, highly lethal fibrin- not been established definitively. ous pleuropneumonia known as contagious caprine pleu- Source: isolated from the nasopharynx of guinea pigs ropneumonia (CCPP) in goats (McMartin et al., 1980). (Hill, 1984). A respiratory tract disease of similar pathology found in DNA G+C content (mol%): 30 (Tm). association with Mycoplasma capricolum subsp. capripneumo- Type strain: 117C, NCTC 11700, ATCC 43016. niae has been reported in sheep, mouflon, and ibex (Arif Sequence accession no. (16S rRNA gene): AF125879. et al., 2007; Shiferaw et al., 2006). Transmission occurs via AL droplet aerosol. 26. Mycoplasma citelli Rose, Tully and Langford 1978, 571 Tetracyclines and tylosin are effective chemotherapeutic ci.tel¢li. N.L. n. Citellus a genus of ground squirrel; N.L. gen. agents; however, treatment of endemic herds is not often n. citelli of Citellus. Genus I. Mycoplasma 593

Cells are highly pleomorphic. Motility for this species DNA G+C content (mol%): 32 (Bd). has not been assessed. Colonies on solid agar have a char- Type strain: 694, ATCC 33549, NCTC 10184. acteristic fried-egg appearance. Grows well in SP-4 medium Sequence accession no. (16S rRNA gene): AF221112. supplemented with glucose at 37°C. Further comment: previously known as avian serovar (sero- No evidence of pathogenicity. Mode of transmission has type) L (Yoder and Hofstad, 1964). not been established definitively. 30. Mycoplasma columbinum Shimizu, Ernø and Nagatomo Source: isolated from the trachea, lung, spleen, and liver 1978, 545AL of ground squirrels (Rose et al., 1978). DNA G+C content (mol%): 27.4 (Bd). co.lum.bi¢num. L. neut. adj. columbinum pertaining to a Type strain: RG-2C, ATCC 29760, NCTC 10181. pigeon. Sequence accession no. (16S rRNA gene): AF412973. Cells are pleomorphic and vary from coccoid to ring forms. Motility for this species has not been assessed. Colo- 27. Mycoplasma cloacale Bradbury and Forrest 1984, 392VP nies on solid medium have typical fried-egg morphology. clo.a.ca¢le. L. neut. adj. cloacale pertaining to a cloaca. Grows in Frey’s medium supplemented with arginine at Cells are primarily spherical. Nonmotile. Colonies on solid 37°C. Produces a “film and spots” reaction. medium exhibit typical fried-egg appearance. Grows well in No evidence of pathogenicity. Mode of transmission has Hayflick medium supplemented with arginine at 37°C. not been assessed definitively. No evidence of pathogenicity. Mode of transmission has Source: isolated from the trachea and oropharynx of feral not been established definitively pigeons and from the brain and lungs of racing pigeons Source: isolated from the cloaca of a turkey; from the lungs, (Bencˇina et al., 1987; Jordan et al., 1981; Keymer et al., trachea, ovaries, and eggs of ducks; and from chickens, 1984; Reece et al., 1986). pheasants, and geese (Bencˇina et al., 1987, 1988; ­Bradbury DNA G+C content (mol%): 27.3 (Bd). et al., 1987; Goldberg et al., 1995; Hinz et al., 1994). Type strain: MMP1, ATCC 29257, NCTC 10178. DNA G+C content (mol%): 26 (Bd). Sequence accession no. (16S rRNA gene): AF221113. Type strain: 383, ATCC 35276, NCTC 10199. 31. Mycoplasma columborale Shimizu, Ernø and Nagatomo Sequence accession no. (16S rRNA gene): AF125592. 1978, 545AL VP 28. Mycoplasma collis Hill 1983b, 849 co.lum.bo.ra¢le. L. n. columba pigeon; L. n. os, oris the mouth; col¢lis. L. gen. n. collis of a hill, alluding to the author who L. neut. suff. -ale suffix used with the sense of pertaining described the species. to; N.L. neut. adj. orale of or pertaining to the mouth; N.L. Cells are primarily coccoidal and are nonmotile. Growth neut. adj. columborale of the pigeon mouth. on a solid medium shows colonies with a typical fried-egg Cells are pleomorphic but predominantly coccoid or appearance. Grows in Hayflick medium supplemented with exhibiting ring forms. Motility for this species has not been glucose at 35–37°C. assessed. Growth on solid medium yields medium to large No evidence of pathogenicity. Mode of transmission has colonies with very small central zones. Grows in Frey’s not been assessed. medium supplemented with glucose at 37°C. Source: isolated from the conjunctivae of captive rats and Pathogenicity for pigeons is unconfirmed, but one report mice (Hill, 1983b). References to isolation of Mycoplasma described airsacculitis in experimentally inoculated chick- collis from domestic dogs appear to have been in error ens. Mode of transmission has not been assessed defini- (Chalker and Brownlie, 2004). tively.

DNA G+C content (mol%): 28 (Tm). Source: isolated from the trachea and oropharynx of feral Type strain: 58B, NCTC 10197, ATCC 35278. pigeons and fantail pigeons; from the oropharynx and Sequence accession no. (16S rRNA gene): AF538681. sinuses of racing pigeons; and from corvids and house flies (Bencˇina et al., 1987; Bradbury et al., 2000; Jordan et al., 29. Mycoplasma columbinasale Jordan, Ernø, Cottew, Hinz and 1981; Kempf et al., 2000; Keymer et al., 1984; MacOwan Stipkovits 1982, 114VP et al., 1981; Nagatomo et al., 1997; Reece et al., 1986). co.lum.bi.na.sa¢le. L. n. columbus a pigeon; L. n. nasus nose; DNA G+C content (mol%): 29.2 (Bd). L. neut. suff. -ale suffix used with the sense of pertaining to; Type strain: MMP4, ATCC 29258, NCTC 10179. N.L. neut. adj. nasale pertaining to the nose; N.L. neut. adj. Sequence accession no. (16S rRNA gene): AF412975. columbinasale pertaining to the nose of a pigeon. 32. Mycoplasma conjunctivae Barile, Del Giudice and Tully Cells are coccoid to coccobacillary. Motility for this 1972, 74AL species has not been assessed. Colonies on solid medium exhibit typical fried-egg appearance. Grows well in SP-4 con.junc.ti¢va.e. N.L. n. conjunctiva the membrane joining the medium supplemented with arginine at 35–37°C. Produces eyeball to the lids; N.L. gen. n. conjunctivae of conjunctiva. a “film and spots” reaction. Cells are coccoid to coccobacillary. Motility for this spe- No evidence of pathogenicity. Mode of transmission has cies has not been assessed. Colonies grown on solid medium not been assessed definitively. may have elevated centers and a greenish, brownish, or Source: isolated from the turbinates of rock pigeons, olive color. Grows well in SP-4 medium supplemented with racing pigeons, and fantail pigeons (Bencˇina et al., 1987; glucose at 37°C. Keymer et al., 1984; Nagatomo et al., 1997; Yoder and Pathogenic; causes infectious keratoconjunctivitis that Hofstad, 1964). can either resolve into a carrier state or result in complete 594 Family I. Mycoplasmataceae

or near-complete blindness in goats, sheep, chamois, and markedly small centers. Grows well in Hayflick broth sup- ibex (Cottew, 1979; Mayer et al., 1997). Mode of transmis- plemented with glucose at 37°C. sion is via direct contact. No evidence of pathogenicity. Mode of transmission has Though tetracyclines are an effective antimicrobial ther- not been established. apy in vivo, treatment to eradicate Mycoplasma conjunctivae Source: isolated from the conjunctivae and nasopharynx from herds is not often attempted as the economic burden of Chinese hamsters (Hill, 1983a). of infection is low. Topical treatment of secondary infec- DNA G+C content (mol%): not determined. tions is often necessary (Slatter, 2001). Several commercial Type strain: CH, NCTC 10190, ATCC 35279. diagnostic assays have been described. Sequence accession no. (16S rRNA gene): AF412976. Source: isolated from the conjunctivae of goats, sheep, 36. Mycoplasma crocodyli Kirchhoff, Mohan, Schmidt, Runge, chamois, and Alpine ibex. Brown, Brown, Foggin, Muvavarirwa, Lehmann and Floss- DNA G+C content (mol%): not determined. dorf 1997, 746VP Type strain: HRC581, ATCC 25834, NCTC 10147. Sequence accession no. (16S rRNA gene): AY816349. cro.co.dy¢li. N.L. n. Crocodylus (from L. n. crocodilus croco- dile) generic name of the crocodile; N.L. gen. n. crocodyli 33. Mycoplasma corogypsi Panangala, Stringfellow, Dybvig, of Crocodylus. Woodard, Sun, Rose and Gresham 1993, 589VP Cells are coccoid. Nonmotile. Colonies on solid medium co.ro.gyp¢si. Gr. n. korax -acos a raven (black); Gr. n. gyps gypos show a typical fried-egg appearance. Grows very rapidly in a vulture; N.L. gen. n. corogypsi (sic) of a raven vulture. SP-4 medium supplemented with glucose at 30°C. Cells are highly pleomorphic and show small circular Pathogenic; causes exudative polyarthritis and rarely budding processes abutting elongated cells. Motility for this pneumonia in crocodiles. The natural mode of transmis- species has not been assessed. Colonies on solid medium sion has not been assessed definitively; however, experimen- have a fried-egg appearance. Grows in Frey’s medium sup- tal infection resulting in the reproduction of disease was plemented with glucose at 37°C. achieved by intracoelomic and/or intrapulmonary inocula- Pathogenicity has not been established, although asso- tion. ciated with abscess formation in a black vulture. Myco- Tetracyclines are effectively used to alleviate clinical signs plasma corogypsi has been isolated from clinically normal in farmed crocodiles. A bacterin vaccine effective at control- captive falcons, and may represent a commensal of this ling infection and preventing disease has been described species. Mode of transmission has not been assessed (Mohan et al., 2001). definitively. Source: isolated from the joints and lungs of Nile croco- Source: isolated from the abscessed footpad of a black vul- diles (Mohan et al., 1997). ture, and from captive falcons (Lierz et al., 2002; Panangala DNA G+C content (mol%): 27.6 (Bd). et al., 1993). Type strain: MP145, ATCC 51981. DNA G+C content (mol%): 28 (Bd). Sequence accession no. (16S rRNA gene): AF412977. Type strain: BV1, ATCC 51148. 37. Mycoplasma cynos Røsendal 1973, 53AL Sequence accession no. (16S rRNA gene): L08054. cy¢nos. Gr. n. cyon, cynos a dog; N.L. gen. n. cynos of a dog. 34. Mycoplasma cottewii DaMassa, Tully, Rose, Pitcher, Leach Cells are coccoid to coccobacillary. Motility for this spe- and Cottew 1994, 483VP cies has not been assessed. Colonies on solid medium have cot.te¢wi.i. N.L. masc. gen. n. cottewii of Cottew, named for defined centers and scalloped perimeters. Grows well in Geoffrey S. Cottew, an Australian veterinarian who was a co- SP-4 medium supplemented with arginine at 37°C. isolator of the organism. Pathogenic; causes pneumonia, bronchitis, and rarely cys- Cells are primarily coccoid. Nonmotile. Formation of titis in domestic dogs. The mode of transmission is via drop- biofilms has been demonstrated (McAuliffe et al., 2006). let aerosol, as demonstrated by studies housing infected Growth on solid medium shows colonies with a typical fried- and sentinel dogs (Røsendal and Vinther, 1977). egg appearance. Grows well in Hayflick medium supple- Source: isolated from the lungs, trachea, nasopharynx, mented with glucose at 37°C. urine, prepuce, prostate, cervix, vagina, and conjunctivae No evidence of pathogenicity. Mode of transmission has of domestic dogs (Chalker, 2005). not been established. DNA G+C content (mol%): 25.8 (Bd). Source: isolated from the external ear canals and rarely Type strain: H 831, ATCC 27544, NCTC 10142. the sinuses of goats (Damassa et al., 1994). Sequence accession no. (16S rRNA gene): AF538682. DNA G+C content (mol%): 27 (T ). m 38. Mycoplasma dispar Gourlay and Leach 1970, 121AL Type strain: VIS, ATCC 51347, NCTC 11732, CIP 105678. Sequence accession no. (16S rRNA gene): U67945. dis¢par. L. neut. adj. dispar dissimilar, different. Cells range from coccoid to short and filamentous. Motil- 35. Mycoplasma cricetuli Hill 1983a, 117VP ity for this species has not been assessed. An extracellular cri.ce.tu¢li. N.L. n. Cricetulus generic name of the Chinese capsule can be visualized by electron microscopy following hamster, Cricetulus griseus; N.L. gen. n. cricetuli of Cricetulus. staining with ruthenium red. Colonies on solid medium Cells are coccoid to pleomorphic. Nonmotile. Colony have a granular, lacy, or reticulated appearance with no or growth on solid medium has a fried-egg appearance with a poorly defined central area. Grows in SP-4 medium or Genus I. Mycoplasma 595

modified Friis medium supplemented with glucose and calf Cells are pleomorphic. Motility for this species has not thymus DNA at 37°C. been assessed. Colonies on solid medium show a typical Pathogenic; causes pneumonia and rarely mastitis in cat- fried-egg appearance. Grows in Hayflick medium supple- tle. Mode of transmission is by droplet aerosol. mented with glucose at 37°C. Source: isolated from the lower respiratory tract and Opportunistic pathogen. Associated with endometri- udders of cattle (Gourlay and Howard, 1979; Hodges et al., tis, vulvitis, balanoposthitis, impaired fecundity, and abor- 1983). tion in horses; however, Mycoplasma equigenitalium is highly

DNA G+C content (mol%): 28.5–29.3 (Tm). prevalent in clinically normal horses (Spergser et al., 2002). Type strain: 462/2, ATCC 27140, NCTC 10125. Mode of transmission is via sexual contact. Sequence accession no. (16S rRNA gene): AF412979. Source: isolated from the cervix, semen, and aborted foals, and rarely from the trachea, of horses (Lemcke, 1979). 39. Mycoplasma edwardii Tully, Barile, Del Giudice, Carski, DNA G+C content (mol%): 31.5 (Bd). Armstrong and Razin 1970, 349AL Type strain: T37, ATCC 29869, NCTC 10176. ed.war¢di.i. N.L. masc. gen. n. edwardii of Edward, named Sequence accession no. (16S rRNA gene): AF221120. after Derrick Graham ff. Edward (1910–1978), who first iso- AL lated this organism. 42. Mycoplasma equirhinis Allam and Lemcke 1975, 405 Cells are coccobacillary to short and filamentous. Motil- e.qui.rhi¢nis. L. n. equus, equi a horse; Gr. n. rhis, rhinos nose; ity for this species has not been assessed. Colonies on solid N.L. gen. n. equirhinis of the nose of a horse. medium show a typical fried-egg appearance. Grows well in Cells are coccoid to coccobacillary. Motility for this spe- SP-4 medium supplemented with glucose at 37°C. cies has not been assessed. Colonies on solid medium show Opportunistic pathogen; commonly found as a commen- a typical fried-egg appearance. Grows in SP-4 or Hayflick sal of the oral and/or nasal cavities and urogenital tract of medium supplemented with arginine at 37°C. domestic dogs. Mycoplasma edwardii is rarely associated with Opportunistic pathogen; associated with rhinitis and pneumonia, arthritis, and septicemia of domestic dogs, often pneumonitis in horses, but can also be found in clinically as a secondary pathogen compounding an existing lesion. normal animals. Mode of transmission is via droplet aero- Mode of transmission has not been established definitively. sol. Source: isolated from the oropharynx, nasopharynx, Source: isolated from the nasopharynx, nasal turbinates, trachea, lungs, prepuce, vagina, cervix, blood, and syn- trachea, tonsils, and semen of horses, and from the ovial fluid of domestic dogs (Chalker, 2005; Stenske et al., nasopharynx of cattle (Lemcke, 1979; Spergser et al., 2002; 2005). ter Laak et al., 1992a).

DNA G+C content (mol%): 29.2 (Tm). DNA G+C content (mol%): not determined. Type strain: PG-24, ATCC 23462, NCTC 10132. Type strain: M432/72, ATCC 29420, NCTC 10148. Sequence accession no. (16S rRNA gene): U73903. Sequence accession no. (16S rRNA gene): AF125585. 40. Mycoplasma elephantis Kirchhoff, Schmidt, Lehmann, 43. Mycoplasma falconis Poveda, Giebel, Flossdorf, Meier and Clark and Hill 1996, 440VP Kirchhoff 1994, 97VP e.le.phan¢tis. L. n. elephas, -antis elephant; L. gen. n. elephan- fal.co¢nis. L. gen. n. falconis of the falcon, the host from tis of the elephant. which the organism was first isolated. Cells are coccoidal. Nonmotile. Colonies on solid Cells are coccoid. Motility for this species has not been medium show a typical fried-egg appearance. Grows well in assessed. Colonies on solid medium have a fried-egg appear- Hayflick medium supplemented with glucose at 37°C. ance. Grows well in modified Frey’s medium supplemented Probable commensal. No pathology was observed at the with arginine at 37°C. site of isolation (i.e., the vagina and urethra); however, Pathogenicity has not been established. Associated with isolation was achieved almost exclusively from arthritic respiratory tract infections of saker falcons, although can animals with evidence of rheumatoid factor. The possibil- also be isolated from clinically normal birds. Mode of trans- ity thus exists that the clinical status of the animals was mission has not been established definitively. due to sexually acquired reactive arthritis, which has been Source: isolated from the trachea of falcons (Lierz et al., observed with other Mycoplasma species known to parasitize 2002, 2008a, b). the urogenital tract (Blanchard and Bébéar, 2002). Mode of DNA G+C content (mol%): 27.5 (Bd). transmission has not been established definitively. Type strain: H/T1, ATCC 51372. Source: isolated from the vagina and urethra of captive Sequence accession no. (16S rRNA gene): AF125591. elephants (Clark et al., 1980, 1978). 44. Mycoplasma fastidiosum Lemcke and Poland 1980, 161VP DNA G+C content (mol%): 24 (Bd). Type strain: E42, ATCC 51980. fas.ti.di.o¢sum. L. neut. adj. fastidiosum fastidious, referring Sequence accession no. (16S rRNA gene): AF221121. to the nutritionally fastidious nature of the organism on pri- mary isolation. 41. Mycoplasma equigenitalium Kirchhoff 1978, 500AL Cells are highly filamentous and filaments are twisted at e.qui.ge.ni.ta¢li.um. L. n. equus, equi the horse; L. pl. n. geni- regular intervals along their length. Helical forms like those talia the genitals; N.L. pl. gen. n. equigenitalium of equine of Spiroplasma species are not produced. Nonmotile. Colo- genitalia. nies on solid medium show a typical fried-egg appearance. 596 Family I. Mycoplasmataceae

Grows in SP-4 or Frey’s medium supplemented with glucose Source: isolated from the oropharynx of domestic cats; at 37°C. from the nasopharynx, lungs, and urogenital tract of domes- No evidence of pathogenicity. Mode of transmission has tic dogs; and from the respiratory tract of horses (Chalker, not been assessed definitively. 2005; Heyward et al., 1969; Lemcke, 1979). Source: isolated from the nasopharynx of horses (Lemcke DNA G+C content (mol%): 29.1 (Bd). and Poland, 1980). Type strain: Ben, ATCC 25749, NCTC 10159. DNA G+C content (mol%): 32.3 (Bd). Sequence accession no. (16S rRNA gene): U16758. Type strain: 4822, NCTC 10180, ATCC 33229. Further comment: this organism was first described during Sequence accession no. (16S rRNA gene): AF125878. a time when the only named genus of mollicutes was Myco- plasma. Its publication coincided with the first proposal of 45. Mycoplasma faucium Freundt, Taylor-Robinson, Purcell, the genus Acholeplasma (Edward and Freundt, 1969, 1970), Chanock and Black 1974, 253AL with which Mycoplasma feliminutum is properly affiliated fau¢ci.um. L. pl. n. fauces, -ium the throat; L. gen. pl. n. fau- through established phenotypic (Heyward et al., 1969) cium of the throat. and 16S rRNA gene sequence (Brown et al., 1995) simi- Cells are coccoidal. Motility for this species has not been larities. This explains the apparent inconsistencies with its assessed. Colonies on solid medium show a typical fried- assignment to the genus Mycoplasma. The name Mycoplasma egg appearance, but are more loosely attached to the agar feliminutum should therefore be revised to Acholeplasma surface than are the colonies of most other mycoplasmas. feliminutum comb. nov. Grows well in SP-4 medium supplemented with arginine at 48. Mycoplasma felis Cole, Golightly and Ward 1967, 1456AL 37°C. Produces a “film and spots” reaction. fe¢lis. L. n. felis a cat, L. gen. n. felis of a cat. Probable commensal. Most commonly found as a com- mensal of the human oropharynx; however, recent isola- Cells are coccobacillary to filamentous. Motility for this tions of Mycoplasma faucium have been made from brain species has not been assessed. Colonies on solid media dis- abscesses (Al Masalma et al., 2009). Mode of transmission play the typical fried-egg morphology. Grows well in SP-4 has not been established definitively. medium supplemented with glucose at 37°C. Source: isolated from the oropharynx and brain of Pathogenic; associated with conjunctivitis, rhinitis, ulcer- humans, and from the oral cavity of numerous species of ative keratitis, and polyarthritis in domestic cats, and upper nonhuman primates (Freundt et al., 1974; Somerson and and lower respiratory tract infection in horses. Mycoplasma Cole, 1979). felis can also be isolated from clinically normal domestic DNA G+C content (mol%): not determined. cats, domestic dogs, and horses. The mode of transmission Type strain: DC-333, ATCC 25293, NCTC 10174. has not been established definitively. Sequence accession no. (16S rRNA gene): AF125590. Source: isolated from the conjunctivae, nasopharynx, lungs, and urogenital tract of domestic cats; from the lungs, 46. Mycoplasma felifaucium Hill 1988, 449VP (Effective publica- tonsils, trachea nasopharynx of horses; from the orophar- tion: Hill 1986, 1927) ynx and trachea of domestic dogs; and from the synovial fe.li.fau¢ci.um. L. n. felis cat; L. pl. n. fauces, -ium throat; N.L. fluid of an immunocompromised human (Lemcke, 1979) gen. pl. n. felifaucium of the feline throat. Røsendal, 1979; (Bonilla et al., 1997; Gray et al., 2005; Cells are primarily coccoidal. Nonmotile. Colonies on Hooper et al., 1985).

solid medium show a typical fried-egg appearance. Grows DNA G+C content (mol%): 25.2 (Tm). well in SP-4 or Hayflick medium supplemented with argin- Type strain: CO, ATCC 23391, NCTC 10160. ine at 37°C. Produces a “film and spots” reaction. Sequence accession no. (16S rRNA gene): U09787. No evidence of pathogenicity. Mode of transmission has Further comment: the proposed species “Mycoplasma not been established definitively. equipharyngis” (Kirchoff, 1974) has been reported in horses. Source: isolated from the oropharynx of captive pumas Further characterization has demonstrated unequivocally (Felis concolor; Hill, 1986). that these isolates are Mycoplasma felis and all mention of

DNA G+C content (mol%): 31 (Tm). “Mycoplasma equipharyngis” should be considered equivalent Type strain: PU, NCTC 11703, ATCC 43428. to Mycoplasma felis (Lemcke, 1979). Sequence accession no. (16S rRNA gene): U15795. 49. Mycoplasma fermentans Edward 1955, 90AL 47. Mycoplasma feliminutum Heyward, Sabry and Dowdle fer.men¢tans. L. part. adj. fermentans fermenting. 1969, 621AL Cells are filamentous. Motility has not been established fe.li.mi.nu¢tum. L. n. felis a cat; L. neut. part. adj. minutum for this species. Colonies on solid media display typical fried- small; N.L. neut. adj. feliminutum a small colony organism egg morphology. Grows well in SP-4 or Hayflick medium isolated from cats. supplemented with either arginine or glucose at 37°C. Morphology is poorly defined. Motility for this species has Pathogenicity unclear; associated with balanitis, vulvovag- not been assessed. Colonies are relatively small and irregu- initis, salpingitis, respiratory distress syndrome, pneumo- lar in shape. Grows well in SP-4 medium supplemented with nia, and development of rheumatoid arthritis. Mycoplasma glucose at 37°C. fermentans has also been tenuously linked with the progres- No evidence of pathogenicity. Mode of transmission has sion of AIDS, chronic fatigue syndrome, Gulf War syn- not been established definitively. drome, Adamantiades-Behçet’s disease, and fibromyalgia. Genus I. Mycoplasma 597

The connection of the preceding clinical syndromes with DNA G+C content (mol%): 28 (Bd). Mycoplasma fermentans is highly equivocal, as different stud- Type strain: DD, ATCC 33550, NCTC 10183. ies have reached markedly different conclusions. Mode of Sequence accession no. (16S rRNA gene): L24104. transmission has not been established definitively. Further comment: previously known as avian serotype D Source: isolated from the urine, urethra, rectum, penis, (Kleckner, 1960). cervix, vagina, fallopian tube, amniotic fluid, blood, synovial 52. Mycoplasma gallinarum Freundt 1955, 73AL fluid, and throat of humans; from the cervix of an African green monkey (Chlorocebus sp.); and from the vagina of a gal.li.na¢rum. L. n. gallina a hen; L. gen. pl. n. gallinarum sheep (Blanchard et al., 1993; Nicholas et al., 1998; Taylor- of hens. Robinson and Furr, 1997; Waites and Talkington, 2005). Cells are coccoid to coccobacillary. Nonmotile. Colo-

DNA G+C content (mol%): 28.7 (Tm) nies on solid medium have a typical fried-egg appearance. Type strain: PG18, ATCC 19989, NCTC 10117, NBRC Grows well in Frey’s medium supplemented with arginine 14854. at 37°C. The organism shares some antigens in immunodif- Sequence accession no. (16S rRNA gene): M24289. fusion tests with Mycoplasma iners, Mycoplasma columbinasale, and Mycoplasma meleagridis. 50. Mycoplasma flocculare Meyling and Friis 1972, 289AL Commensal of gallinaceous birds; little evidence exists floc.cu.la¢re. L. dim. n. flocculus a small flock or tuft of wool; for the pathogenicity of isolates in such hosts. Mycoplasma L. neut. suff. -are suffix denoting pertaining to; N.L. neut. gallinarum may have a role in airsacculitis of geese and par- adj. flocculare resembling a small floc of wool, referring to ticipate in complex infection of chickens. Mode of transmis- the tendency of the organism to form clumps of flocculent sion has not been assessed definitively. material in broth culture. Source: isolated from the respiratory tract of chickens, tur- Cells are coccoid to coccobacillary. Motility for this species keys, ducks, geese, red jungle fowl, bamboo partridge, spar- has not been assessed. Colonies on solid media are slightly row, swan, and demoisella crane; and from sheep (Kisary convex with a coarsely granular surface and lack a defined et al., 1976; Kleven et al., 1978; Shimizu et al., 1979; Singh center. Aggregates of cells may be produced during growth and Uppal, 1987).

in broth, appearing as small floccular elements upon gentle DNA G+C content (mol%): 26.5–28.0 (Tm, Bd). shaking of the culture. Grows slowly in Friis medium at 37°C. Type strain: PG16, ATCC 19708, NCTC 10120. Opportunistic pathogen; normally regarded as a com- Sequence accession no. (16S rRNA gene): L24105. mensal of the nasopharynx that can cause pneumonia in Further comment: previously known as avian serotype B association with other pathogens, most notably Mycoplasma (Kleckner, 1960). hyopneumoniae. Mode of transmission is via droplet aerosol. 53. Mycoplasma gallisepticum Edward and Kanarek 1960, Mycoplasma flocculare reportedly shares surface antigens 699AL with Mycoplasma hyopneumoniae, potentially complicating serology-based diagnosis of infection (Whittlestone, 1979). gal.li.sep¢ti.cum. L. n. gallus rooster, chicken; L. adj. septi- Source: isolated from the nasopharynx, lungs, pericar- cus -a -um producing a putrefaction, putrefying, septic; N.L. dium, and conjunctivae of pigs. neut. adj. gallisepticum hen-putrefying (infecting). DNA G+C content (mol%): 33 (Bd). Cells are coccoid, ovoid, and elongated pear-shaped with Type strain: Ms42, ATCC 27399, NCTC 10143. a highly structured polar body, called the bleb. Cells are Sequence accession no. (16S rRNA gene): L22210. motile and glide in the direction of the terminal bleb. Glid- ing speed varies among strains. Colonies on solid medium 51. Mycoplasma gallinaceum Jordan, Ernø, Cottew, Hinz and may be small and not necessarily of typical fried-egg appear- Stipkovits 1982, 114VP ance. Grows well in SP-4 or Hayflick medium supplemented gal.li.na¢ce.um. L. neut. adj. gallinaceum pertaining to a with glucose at 37°C (Balish and Krause, 2006; Hatchel domestic fowl. et al., 2006; Nakane and Miyata, 2009). Cells are coccoid to coccobacillary. Motility for this spe- Pathogenic. Causes a characteristic combination of cies has not been assessed. Colonies on solid medium have pneumonia, tracheitis, and airsacculitis (collectively typical fried-egg morphology although some are devoid of termed chronic respiratory disease); salpingitis and atro- a central core. Grows well in Frey’s medium supplemented phy of the ovaries, isthmus, and cloaca resulting in poor with glucose at 37°C. egg quality and reduced hatchability; arthritis or synovitis; Opportunistic pathogen associated with tracheitis, air- and keratoconjunctivitis in chickens; infectious sinusitis, sacculitis, or conjunctivitis in chickens, turkeys, ducks, and coryza, airsacculitis, arthritis or synovitis, encephalitis, pheasants. Mycoplasma gallinaceum has been reported to meningitis, ataxia, and torticollis in turkeys; conjunctivi- complicate cases of infectious synovitis due to Mycoplasma tis and coryza featuring a high mortality rate in finches synoviae in chickens. The mode of transmission has not and grosbeaks; and respiratory disease in additional game been assessed definitively. birds including quail, partridges, pheasants, and pea- Source: isolated from upper and lower respiratory tract of fowl. Lesions established by Mycoplasma gallisepticum are chickens, turkeys, pheasants, partridges, and ducks; from often complicated by additional avian pathogens includ- the conjunctivae of pheasants; and from the synovial fluid ing Mycoplasma synoviae, avian strains of Escherichia coli, of chickens (Bradbury et al., 2001; Tiong, 1990; Welchman Newcastle disease virus, and infectious bronchitis virus. et al., 2002; Yagihashi et al., 1983). Established mechanisms of transmission include droplet 598 Family I. Mycoplasmataceae

aerosols, direct contact with infected animals or fomites, DNA G+C content (mol%): 27 (Bd). and vertical transmission. Type strain: WR1, ATCC 33551, NCTC 10186. Tetracyclines, macrolides, aminoglycosides, fluoroqui- Sequence accession no. (16S rRNA gene): AF412980. nolones, and pleuromutilins are effective chemotherapeu- Further comment: previously known as avian serotype F tic agents; however, treatment is typically only sought for (Kleckner, 1960). individual birds, as medicating a commercial flock is not 55. Mycoplasma gateae Cole, Golightly and Ward 1967, 1456AL considered an effective control strategy. Vaccination and management strategies (i.e., single age “all in/all out” sys- ga.te¢ae. N.L. gen. n. gateae (probably from Spanish gato, a tems and culling of endemic flocks) are more commonly cat) of a cat. utilized. Multiple live and killed vaccines are commercially Morphology is poorly defined. Motility for this species available, but suffer from residual pathogenicity, the need has not been assessed. Colonies on solid medium are vacu- to develop a carrier state to provide protective immunity, olated and lack a well-defined central spot. Grows well in adverse reactions, or low efficacy. Experimental vaccines SP-4 medium at 37°C. have also been described. Mycoplasma gallisepticum shares Opportunistic pathogen; can cause polyarthritis in surface antigens with Mycoplasma imitans and Mycoplasma domestic cats (Moise et al., 1983), but appears to be pri- synoviae, potentially complicating serology-based diagnosis marily a commensal species of the oral cavity. Mycoplasma of infection. Numerous molecular diagnostics have been gateae also appears to be a commensal species of domestic described. This organism is listed in the Terrestrial Animal dogs and cattle. Mode of transmission has not been assessed Health Code of the Office International des Epizooties definitively. (http://oie.int; Yogev et al., 1989; Kempf, 1998; Markham Source: isolated from the synovial membrane, orophar- et al., 1999; Gautier-Bouchardon et al., 2002; Ferguson et al., ynx, saliva, and urogenital tract of domestic cats; from the 2004; Browning et al., 2005; Crespo and McMillan, 2008; lungs, oropharynx, trachea, and urogenital tract of domes- Gates et al., 2008; Gerchman et al., 2008; Kleven, 2008). tic dogs; and from the urogenital tract of cattle (Chalker, Source: isolated from the trachea, lungs, air sacs, ovaries, 2005; Gourlay and Howard, 1979; Røsendal, 1979).

oviducts, brain, arterial walls, synovial membranes, synovial DNA G+C content (mol%): 28.5 (Tm). fluid, conjunctivae, and eggs of chickens; from the infraor- Type strain: CS, ATCC 23392, NCTC 10161. bital sinuses, air sacs, brain, meninges, conjunctivae, syn- Sequence accession no. (16S rRNA gene): U15796. ovial membranes, and synovial fluid of turkeys; from the Further comment: the original specific epithetgateae “ ”, conjunctivae, infraorbital sinuses, and trachea of finches; which has been perpetuated in lists of bacterial names and from the respiratory tract of quail, partridges, pheas- approved by the International Committee on Systematics of ants, peafowl, ducks, grosbeak, crows, robins, and blue jays Prokaryotes, the American Type Culture Collection’s Cata- (Bencˇina et al., 2003, 1988; Bradbury and Morrow, 2008; log of Bacteria and Bacteriophages, and in GenBank, is illegiti- Bradbury et al., 2001; Dhondt et al., 2005, 2007; Levisohn mate because the genitive of the medieval Latin word gata and Kleven, 2000; Ley et al., 1996; Mikaelian et al., 2001; (female cat) would have been gatae and there is no word for Murakami et al., 2002; Nolan et al., 2004; Nunoya et al., which gateae would have been a legitimate genitive (Brown 1995; Welchman et al., 2002; Wellehan et al., 2001). et al., 1995). DNA G+C content (mol%): 31.8 (T ), 31 (strain R com- m 56. Mycoplasma genitalium Tully, Taylor-Robinson, Rose, Cole plete genome sequence). and Bové 1983, 395VP Type strain: PG31, X95, ATCC 19610, NCTC 10115. Sequence accession nos: M22441 (16S rRNA gene of ge.ni.ta¢li.um. L. pl. n. genitalia, -ium the genitals; L. gen. pl. strain A5969), NC_004829 (strain R complete genome n. genitalium of the genitals. sequence). Cells are predominantly flask-shaped with a terminal Further comment: previously known as avian serotype A organelle protruding from the cell pole that is narrower (Kleckner, 1960). than that of Mycoplasma gallisepticum and shorter than that of Mycoplasma pneumoniae. The leading end of the terminal 54. Mycoplasma gallopavonis Jordan, Ernø, Cottew, Hinz and structure is often curved. Cells exhibit gliding motility in Stipkovits 1982, 114VP circular patterns and glide in the direction of the terminal gal.lo.pa.vo¢nis. N.L. n. gallopavo, -onis a turkey (Meleagris organelle’s curvature (Hatchel and Balish, 2008). Colonies gallopavo); N.L. gen. n. gallopavonis of a turkey. on solid media are round and possess a defined center that Cells are coccoid to coccobacillary. Motility has not been is somewhat less distinct than most mycoplasma species. assessed for this species. Colonies on solid medium have Grows well in SP-4 medium supplemented with glucose at typical fried-egg morphology. Grows well in Frey’s medium 37°C. supplemented with glucose at 37°C. Pathogenic; causes urethritis, cervicitis, endometritis, Opportunistic pathogen; occasionally associated with and pelvic inflammatory disease. Mycoplasma genitalium is airsacculitis in turkeys, but is also isolated from clinically associated with infertility in humans. Mode of transmis- normal turkeys. Mode of transmission has not been assessed sion is via sexual contact, congenitally, and possibly, in rare definitively. instances, via droplet aerosol. Source: isolated from the choanae, trachea, and air sacs of Macrolides and fluoroquinolones are effective chemo- domestic and wild turkeys (Bencˇina et al., 1987; Cobb et al., therapeutic agents; however, reports indicate that treat- 1992; Hoffman et al., 1997; Luttrell et al., 1992). ment should be extensive, as clinical signs and detection Genus I. Mycoplasma 599

of Mycoplasma genitalium tend to recur following cessation. 59. Mycoplasma haemocanis (Kikuth 1928) Messick, Walker, This is potentially due to sequestration within host cells. Raphael, Berent and Shi 2002, 697VP [Bartonella canis Kikuth Mycoplasma genitalium shares numerous surface antigens 1928, 1730; Haemobartonella (Bartonella) canis (Kikuth 1928) with Mycoplasma pneumoniae, complicating serology-based Tyzzer and Weinman 1939, 151; Kreier and Ristic 1984, 726] diagnosis of infection. Numerous molecular diagnostics ha.e.mo.ca¢nis Gr. neut. n. haema blood; L. fem. gen. n. canis have been described, but few have been developed com- of the dog; N.L. gen. n. haemocanis of dog blood. mercially (Jensen, 2004; Waites and Talkington, 2005). Cells are coccoid to pleomorphic. Motility for this species Source: isolated or detected in the urogenital tract, urine, has not been assessed. The morphology of infected erythro- rectum, synovial fluid, conjunctiva, and nasopharynx of cytes is altered, demonstrating a marked depression at the humans (Baseman et al., 1988; de Barbeyrac et al., 1993; site of Mycoplasma haemocanis attachment. This species has Jensen, 2004; Waites and Talkington, 2005). not been grown on any artificial medium; therefore, nota- DNA G+C content (mol%): 32.4 (Bd), 31 (strain G-37T com- ble biochemical parameters are not known. plete genome sequence). Pathogenic; causes hemolytic anemia in domestic dogs. Type strain: G-37, ATCC 33530, CIP 103767, NCTC Transmission is vector-borne and mediated by the brown 10195. dog tick (Rhipicephalus sanguineus). Sequence accession nos: X77334 (16S rRNA gene), Source: observed in association with erythrocytes of NC_000908 (strain G-37T complete genome sequence), domestic dogs (Hoskins, 1991). CP000925 (strain JCVI-1.0 complete genome sequence). DNA G+C content (mol%): not determined. 57. Mycoplasma glycophilum Forrest and Bradbury 1984, 355VP Type strain: not established. (Effective publication: Forrest and Bradbury 1984, 602) Sequence accession no. (16S rRNA gene): AF197337. gly.co.phi¢lum. Gr. adj. glykys sweet (this adjective was used 60. Mycoplasma haemofelis (Clark 1942) Neimark, ­Johansson, to coin the noun glucose); N.L. neut. adj. philum (from Gr. Rikihisa and Tully 2002b, 683VP [Eperythrozoon felis Clark neut. adj. philon) friend, loving; N.L. neut. adj. glycophilum 1942, 16; Haemobartonella felis (Clark 1942) Flint and sweet-loving, intended to mean glucose-loving. ­McKelvie 1956, 240 and Kreier and Ristic 1984, 725] Cells are spherical or elliptical with an extracellular ha.e.mo.fe¢lis. Gr. neut. n. haema blood; L. fem. gen. n. felis layer. Nonmotile. Growth on solid medium shows colonies of the cat; N.L. gen. n. haemofelis of cat blood. with typical fried-egg appearance. Grows well in Hayflick medium supplemented with glucose at 37°C. Cells are coccoid. Motility for this species has not been Pathogenicity has not been established, although there assessed. This species has not been grown on artificial may be an association with a slight decrease in hatchability. medium; therefore, notable biochemical parameters are Mode of transmission has not been assessed definitively. not known. Source: isolated from the respiratory tract and cloaca of Pathogenic; causes hemolytic anemia in cats. The mode chickens; and from the respiratory tract of turkeys, pheas- of transmission is percutaneous or oral; an insect vector has ants, partridges, ducks, and geese (Forrest and Bradbury, not been identified, although fleas have been implicated 1984). (Woods et al., 2005). DNA G+C content (mol%): 27.5 (Bd). Tetracyclines and fluoroquinolones are effective thera- Type strain: 486, ATCC 35277, NCTC 10194. peutic agents (Dowers et al., 2002; Tasker et al., 2006). Sequence accession no. (16S rRNA gene): AF412981. Source: observed in association with erythrocytes of domestic cats. 58. Mycoplasma gypis Poveda, Giebel, Flossdorf, Meier and DNA G+C content (mol%): 38.5–38.8 (genome sequence Kirchhoff 1994, 98VP survey of strain OH; Berent and Messick, 2003; J.B. Messick gy¢pis. Gr. n. gyps, gypos vulture; N.L. gen. n. gypis of the vul- et al., unpublished). ture, the host from which the organism was first isolated. Type strain: not established Cells are coccoid or round. Motility for this species has Sequence accession no. (16S rRNA gene): U88563. not been assessed. Colonies on solid medium have a fried- 61. Mycoplasma haemomuris (Mayer 1921) Neimark, Johans- egg appearance. Grows in Frey’s medium supplemented son, Rikihisa and Tully 2002b, 683VP (Bartonella muris Mayer with arginine at 37°C. Produces a “film and spots” reac- 1921, 151; Bartonella muris ratti Regendanz and Kikuth 1928, tion. 1578; Haemobartonella muris Tyzzer and Weinman 1939, Pathogenicity has not been established. Associated with 143) respiratory tract disease of griffon vultures, but has also been isolated from healthy birds of prey. Mode of transmis- ha.e.mo.mu¢ris. Gr. neut. n. haema blood; L. masc. gen. n. sion has not been assessed definitively. muris of the mouse; N.L. gen. n. haemomuris of mouse blood. Source: isolated from the trachea of griffon vultures (Grif- Cells are coccoid and some display dense inclusion par- fon fulvus), and from the trachea and air sacs of Eurasian ticles. Motility for this species has not been assessed. The buzzards, red kites, and Western marsh harriers (Lierz morphology of infected erythrocytes is altered, demonstrat- et al., 2008a, 2000; Poveda et al., 1994). ing a marked depression at the site of Mycoplasma haemo- DNA G+C content (mol%): 27.1 (Bd). muris attachment. This species has not been grown on any Type strain: B1/T1, ATCC 51370. artificial medium; therefore, notable biochemical para­ Sequence accession no. (16S rRNA gene): AF125589. meters are not known. 600 Family I. Mycoplasmataceae

Opportunistic pathogen; causes anemia in splenectomized medium supplemented with arginine at 37°C and produces or otherwise immunosuppressed mice. Transmission is vector- a “film and spots” reaction. borne and mediated by the rat louse (Polypax spinulosa). No evidence of pathogenicity. Mode of transmission has Source: observed in association with erythrocytes of wild not been established definitively. and captive mice, and hamsters. Source: isolated from the nasopharynx of pigs (Erickson DNA G+C content (mol%): not determined. et al., 1986).

Type strain: not established. DNA G+C content (mol%): 24 (Bd, Tm). Sequence accession no. (16S rRNA gene): U82963. Type strain: H3-6B F, ATCC 51909, NCTC 11705. Sequence accession no. (16S rRNA gene): U58997. 62. Mycoplasma hominis (Freundt 1953) Edward 1955, 90AL (Micromyces hominis Freundt 1953, 471) 64. Mycoplasma hyopneumoniae (Goodwin, Pomeroy and ho¢mi.nis. L. n. homo, -inis man; L. gen. n. hominis of man. Whittlestone 1965) Maré and Switzer 1965, 841AL (Myco- plasma suipneumoniae Goodwin, Pomeroy and Whittlestone Cells are coccoid to filamentous. Motility for this spe- 1965, 1249) cies has not been assessed. Colonies on solid media have a typical fried-egg appearance. Grows well in SP-4 medium hy.o.pneu.mo¢ni.ae. Gr. n. hys, hyos a swine; Gr. n. pneumonia supplemented with arginine at 37°C. pneumonia; N.L. gen. n. hyopneumoniae of swine pneumonia. Pathogenic; causes pyelonephritis, pelvic inflamma- Cells are coccoid to coccobacillary. Nonmotile. Colo- tory disease, chorioamnionitis, and postpartum fevers in nies on solid medium are very small, lack a defined cen- women; congenital pneumonia, meningitis, and abscesses tral region, and are usually convex with a granular surface. in newborns; and rarely extragenital pathologies including Grows very slowly in modified Friis medium, medium A26, bacteremia, arthritis, osteomyelitis, abscesses and wound and modified SP-4 medium supplemented with glucose at infections, mediastinitis, pneumonia, peritonitis, pros- 37°C. Produces a “film and spots” reaction. thetic- and catheter-associated infections, and infection Pathogenic; causes a very characteristic chronic pneu- of hematomas. Extragenital manifestations of Mycoplasma monitis associated with ciliostasis and marked sloughing hominis infection are more commonly seen in immunosup- of the epithelial lining in pigs. This collection of lesions in pressed individuals, but can be seen in immunocompetent conjunction with Mycoplasma hyopneumoniae is referred to as patients as well. Synergism between Mycoplasma hominis and enzootic pneumonia of pigs (EPP), and is associated with Trichomonas vaginalis infections has been reported and a high morbidity and poor feed conversion (with propor- recent report documents the intraprotozooal location and tional economic loss). The mechanism of transmission is transmission of Mycoplasma hominis with Trichomonas vagi- via droplet aerosol. nalis (Dessi et al., 2006; Germain et al., 1994; Vancini and Tetracyclines and tylosin do not typically eradicate Benchimol, 2008). Mode of transmission is via sexual con- Mycoplasma hyopneumoniae from infected animals, but are tact, congenitally, or by artificial introduction on foreign effective in limiting sequelae. Maintenance of pigs on anti- objects (e.g., catheters) or transplanted tissues. biotics in conjunction with management practices involv- Macrolides and fluoroquinolones are effective chemo- ing adequate nutrition, air quality, and stress reduction therapeutic agents. Combination therapy with metronida- are commonly employed to control the effects of disease zole is required for complex infections involving Trichomonas in endemic herds. Many molecular diagnostics have been vaginalis. Many commercial diagnostics are available for described (Dubosson et al., 2004). Serological diagnostic routine clinical use. techniques utilizing monoclonal antibodies have proven Source: isolated from the urogenital tract, amniotic fluid, successful at distinguishing Mycoplasma hyopneumoniae from placenta, umbilical cord blood, urine, semen, bloodstream, Mycoplasma flocculare, though the two share surface antigens cerebrospinal fluid, synovial fluid, bronchoalveolar lavage (Armstrong et al., 1987). Numerous experimental vaccines fluid, peritoneal aspirates, conjunctivae, bone abscesses, have been described and several commercial vaccines are and hematoma aspirates of humans; and from several available. The latter appear to reduce or eliminate clinical species of nonhuman primates (Somerson and Cole, signs rather than prevent infection (Browning et al., 2005). 1979; Taylor-Robinson and McCormack, 1979; Waites and Source: isolated from the lungs, nasopharynx, tonsils, tra- Talkington, 2005). chea, and bronchiolar lavage fluid of pigs (Marois et al., DNA G+C content (mol%): 33.7 (Tm) 2007; Whittlestone, 1979). Type strain: PG21, ATCC 23114, NCTC 10111, CIP 103715, DNA G+C content (mol%): 27.5 (Bd), 28 (strain JT and NBRC 14850. strain 7448 complete genome sequence), 28.6 (strain 232 Sequence accession no. (16S rRNA gene): M24473. complete genome sequence). 63. Mycoplasma hyopharyngis Erickson, Ross, Rose, Tully and Type strain: J, ATCC 25934, NCTC 10110. Bové 1986, 58VP Sequence accession nos: AY737012 (16S rRNA gene), AE017243 (strain JT complete genome sequence), AE017244 hy.o.pha.ryn¢gis. Gr. n. hys, hyos a swine; N.L. n. pharynx (strain 7448 complete genome sequence), AE017332 (strain -yngis (from Gr. n. pharugx, pharuggos throat) throat; N.L. 232 complete genome sequence). gen. n. hyopharyngis of a hog’s throat. AL Cells are pleomorphic. Motility for this species has not 65. Mycoplasma hyorhinis Switzer 1955, 544 been assessed. Colonies on solid medium have a typical hy.o.rhi¢nis. Gr. n. hys, hyos a swine; Gr. n. rhis, rhinos nose; fried-egg appearance. Grows in medium D-TS or Hayflick N.L. gen. n. hyorhinis of a hog’s nose. Genus I. Mycoplasma 601

Cells are coccoid to coccobacillary. Motility for this species Source: isolated from vertebral abscesses of green has not been assessed. Colonies on solid media display typi- ­iguanas. cal fried-egg morphology. Grows well on S-4 supplemented DNA G+C content (mol%): not determined. with glucose at 37°C. Type strain: 2327, ATCC BAA-1050, NCTC 11745. Mycoplasma hyorhinis is associated with contamination Sequence accession no. (partial 16S rRNA gene sequence): of eukaryotic cell culture and can be removed by treat- AY714305. ment of cells with antibiotics and/or maintenance of cell 68. Mycoplasma imitans Bradbury, Abdul-Wahab, Yavari, lines in antibiotic-containing medium. The noted effects Dupiellet and Bové 1993, 726VP of Mycoplasma hyorhinis on cell-cycle regulation make the i¢mi.tans. L. part. adj. imitans imitating, mimicking, refer- detection and elimination of this organism particularly per- ring to the organism’s phenotypic resemblance to Myco- tinent (Goodison et al., 2007; Schmidhauser et al., 1990). plasma gallisepticum. The most effective classes of antibiotics for cell culture eradication are tetracyclines and fluoroquinolones (Borup- Cells are oval and flask-shaped with a short, wide attach- ­Christensen et al., 1988; Schmitt et al., 1988). ment organelle. Cells are motile and exhibit gliding motil- Pathogenic; associated with arthritis, polyserositis, and ity in the direction of the attachment organelle (Hatchel otitis media in pigs. Mycoplasma hyorhinis is also regarded and Balish, 2008). Colonies have typical fried-egg morphol- as a commensal of the nasopharynx that can occasionally ogy on solid medium. Grows well in SP-4 medium supple- cause pneumonia, often in association with other patho- mented with glucose at 37°C. gens (most notably Mycoplasma hyopneumoniae and Bordetella Pathogenic; causes sinusitis in ducks, geese, and par- bronchiseptica). Mode of transmission is via droplet aerosol. tridges. Disease has been reproduced experimentally. Mode Source: isolated from the nasopharynx, lungs, ear canal, of transmission has not been assessed definitively. synovial fluid, serous cavity, and pericardium of pigs (Friis Diagnosis of infection is potentially complicated by and Szancer, 1994; Ross, 1992; Whittlestone, 1979). numerous factors. Serological cross-reactions occur with Mycoplasma gallisepticum due to known epitopes including DNA G+C content (mol%): 27.8 (Tm). Type strain: BTS-7, ATCC 17981, NCTC 10130, CIP PvpA, the VlhA hemagglutinins, pyruvate dehydrogenase, 104968, NBRC 14858. lactate dehydrogenase, and elongation factor Tu (Jan et al., Sequence accession no. (16S rRNA gene): M24658. 2001; Lavricˇ et al., 2005; Markham et al., 1999; Rosengarten et al., 1995). In addition, the 16S rRNA genes share 99.9% AL 66. Mycoplasma hyosynoviae Ross and Karmon 1970, 710 identity, despite whole genome hybridization showing a hy.o.sy.no.vi¢ae. Gr. n. hys, hyos a swine; N.L. n. synovia fluid relatedness of only 40–46%, potentially complicating molec- in the joints; N.L. gen. n. hyosynoviae of joint fluid of swine. ular diagnostics based on the 16S rRNA gene. Two molecu- Cells are coccoid to filamentous. Motility of this species lar methods for distinguishing these two species have been has not been assessed. Colonies display a typical fried-egg described (Harasawa et al., 2004; Marois et al., 2001). appearance at 37°C. Grows well in SP-4 medium supple- Source: isolated from the nasal turbinates and sinuses of mented with glucose at 37°C. A granular deposit and a waxy ducks, geese, and partridges (Dupiellet, 1984; Ganapathy surface pellicle are produced during growth in broth. and Bradbury, 1998; Kleven, 2003). Pathogenic; causes infectious synovitis, arthritis, and DNA G+C content (mol%): 31.9 (Bd). rarely pericarditis in pigs. Transmission occurs from sows to Type strain: 4229, NCTC 11733, ATCC 51306. piglets or between adults via aerosol. Sequence accession no. (16S rRNA gene): L24103. Lincosamides, fluoroquinolones, and macrolides have 69. Mycoplasma indiense Hill 1993, 39VP been used effectively for treatment in conjunction with in.di.en¢se. N.L. neut. adj. indiense pertaining to India improved disinfection and quarantine during husbandry. (source of the infected primates). Source: isolated from the synovial fluid, nasopharynx, ton- sils, lymph nodes, and pericardium of pigs (Whittlestone, Cells are pleomorphic. Nonmotile. Colonies on agar 1979). have a characteristic fried-egg appearance. Grows well in DNA G+C content (mol%): 28.0 (Bd). SP-4 medium supplemented with arginine at 37°C. Type strain: S16, ATCC 25591, NCTC 10167. No evidence of pathogenicity. Mode of transmission has Sequence accession no. (16S rRNA gene): U26730. not been established. Source: isolated from the throats of a rhesus monkey and 67. Mycoplasma iguanae Brown, Demcovitz, Plourdé, Potter, a baboon (Hill, 1993). VP Hunt, Jones and Rotstein 2006, 763 DNA G+C content (mol%): 32 (Bd). i.gua¢nae. N.L. gen. n. iguanae of the iguana lizard. Type strain: 3T, NCTC 11728, ATCC 51125. Cells are predominantly coccoid. Nonmotile. Colonies on Sequence accession no. (16S rRNA gene): AF125593. solid medium exhibit variable (convex to umbonate) forms; 70. Mycoplasma iners Edward and Kanarek 1960, 699AL mature colonies display sectored centers. Grows well in SP-4 i¢ners. L. neut. adj. iners inactive, inert. medium supplemented with glucose between 25 and 42°C. Associated with pathologic lesions, but unable to repro- The cell morphology is poorly defined. Motility for this duce disease following experimental inoculation (Brown species has not been assessed. Colonies on solid medium are et al., 2007). Mechanism of transmission has not been estab- relatively small and of a typical fried-egg appearance. Grows lished. in Frey’s medium supplemented with arginine at 37°C. 602 Family I. Mycoplasmataceae

No evidence of pathogenicity. Mode of transmission has Type strain: 12MS, ATCC 700289, CIP 105489, DSM not been assessed definitively. 22062. Source: isolated from the respiratory tract of chickens, tur- Sequence accession no. (16S rRNA gene): AF412983. keys, geese pigeons, pheasants, and partridges; and from 73. Mycoplasma leachii Manso-Silván, Vilei, Sachse, Djordjevic, tissues of swine (Bencˇina et al., 1987; Bradbury et al., 2001; Thiaucourt and Frey 2009, 1356VP Taylor-Robinson and Dinter, 1968). le.a.chi¢i. N.L. masc. gen. n. leachii of Leach, named in DNA G+C content (mol%): 29.1 (Tm), 29.6 (Bd). Type strain: PG30, ATCC 19705, NCTC 10165. honor of Dr R.H. Leach, who first characterized this taxon. Sequence accession no. (16S rRNA gene): AF221114. Cells are pleomorphic. Nonmotile. Colonies on solid agar Further comment: previously known as avian serotype E have a characteristic fried-egg appearance. Grows in modi- (Kleckner, 1960). fied Hayflick medium supplemented with glucose at 37°C. Pathogenic; causes polyarthritis, mastitis, abortion, and 71. Mycoplasma iowae Jordan, Ernø, Cottew, Hinz and Stipko- pneumonia in cattle. Mode of transmission has not been vits 1982, 114VP established definitively. i.o¢wa.e. N.L. gen. n. iowae of Iowa. Tetracyclines appear to control infection during acute Cells are pleomorphic and some display a terminal outbreaks (Hum et al., 2000). Mycoplasma leachii shares ­protrusion with possible attachment properties (Gallagher surface antigens with Mycoplasma mycoides subsp. mycoides, and Rhoades, 1983; Mirsalimi et al., 1989). Motile. Colonies Mycoplasma capricolum subsp. capripneumoniae, and Myco- on agar show typical fried-egg appearance. Grows well in plasma capricolum subsp. capricolum, potentially compound- SP-4 medium supplemented with either glucose or arginine ing serology-based diagnosis of infection. at 41–43°C (Grau et al., 1991; Yoder and Hofstad, 1964). Source: isolated from the synovial fluid, udders, expelled Pathogenic; causes airsacculitis and embryo lethality milk, lungs, lymph nodes, pericardium, cervix, vagina, pre- resulting in reduced hatchability in turkeys. Transmission puce, semen, and aborted calves of cattle (Alexander et al., occurs vertically and by direct contact. 1985; Gourlay and Howard, 1979; Hum et al., 2000). Tetracyclines, macrolides, and fluoroquinolones are DNA G+C content (mol%): not determined. effective chemotherapeutic agents; however, medicating Type strain: PG50, NCTC 10133, DSM 21131. a commercial flock is not considered an effective control Sequence accession no. (16S rRNA gene): AF261730. strategy. Management tactics are more commonly uti- Further comment: the assignment of strains formerly called lized. Molecular diagnostic methods have been described “Mycoplasma species bovine group 7 of Leach” to the species (Ramírez et al., 2008; Raviv and Kleven, 2009). Mycoplasma Mycoplasma leachii came in response to a request from the Sub- iowae strains show considerable intra-species antigenic committee on the Taxonomy of Mollicutes of the International heterogeneity and a cross-reactive epitope with both Myco- Committee on Systematics of Prokaryotes for a proposal for plasma gallisepticum and Mycoplasma imitans potentially com- an emended taxonomy for the members of the Mycoplasma plicating serology-based diagnosis of infection (Al-Ankari mycoides phylogenetic cluster (Manso-Silván et al., 2009). and Bradbury, 1996; Dierks et al., 1967; Rosengarten et al., 74. Mycoplasma leonicaptivi corrig. Hill 1992, 521VP 1995). Source: isolated from the air sacs, intestinal tract, and eggs le.o.ni.cap¢ti.vi. L. n. leo, -onis the lion; L. adj. captivus cap- of turkeys, and from the seed of an apple tree with apple tive; N.L. gen. n. leonicaptivi of the captive lion. proliferation disease (Bradbury and Kleven, 2008; Grau Cells are pleomorphic (primarily coccoid). Nonmotile. et al., 1991; Mirsalimi et al., 1989). Colonies on solid medium have a typical fried-egg appear- DNA G+C content (mol%): 25 (Bd). ance. Growth in SP-4 broth supplemented with glucose Type strain: 695, ATCC 33552, NCTC 10185. occurs between 35 and 37°C. Sequence accession no. (16S rRNA gene): M24293. No evidence of pathogenicity. Mode of transmission has Further comment: previously known as avian serotype I not been established. (Yoder and Hofstad, 1964). Source: isolated from the throat and respiratory tract of captive lions and leopards (Hill, 1992). 72. Mycoplasma lagogenitalium Kobayashi, Runge, Schmidt, DNA G+C content (mol%): 27 (Bd). Kubo, Yamamoto and Kirchhoff 1997, 1211VP Type strain: 3L2, NCTC 11726, ATCC 49890. la.go.ge.ni.ta¢li.um. Gr. masc. n. lagos hare; L. neut. pl. gen. Sequence accession no. (16S rRNA gene): U16759. n. genitalium of genitals; N.L. gen. pl. n. lagogenitalium of Further comment: the original spelling of the specific epi- hare’s genitals. thet, leocaptivus (sic), has been corrected by Trüper and Cells are primarily coccoid. Nonmotile. Colonies on agar De’Clari (1998). have a characteristic fried-egg appearance. Grows well in 75. Mycoplasma leopharyngis Hill 1992, 521VP SP-4 medium supplemented with glucose at 37°C. No evidence of pathogenicity. Mechanism of transmis- le.o.pha.ryn¢gis. L. masc. n. leo, -onis lion; N.L. n. pharynx, sion has not been established. -yngis (from Gr. n. pharugx, pharuggos throat) throat; N.L. Source: isolated from the preputial smegma of Afghan gen. n. leopharyngis (sic) of the throat of a lion. pikas (Ochotona rufescens; Kobayashi et al., 1997). Cells are coccoid to pleomorphic. Nonmotile. Colonies

DNA G+C content (mol%): 23 (Tm). on solid medium have a typical fried-egg appearance under Genus I. Mycoplasma 603

anaerobic conditions. Grows in SP-4 broth supplemented 79. Mycoplasma maculosum Edward 1955, 90AL with glucose at optimum temperatures of 35–37°C. Grows ma.cu.lo¢sum. L. neut. adj. maculosum spotted, alluding to a well under both aerobic and anaerobic conditions. crinkled film covering the colonies and spreading between No evidence of pathogenicity. Mechanism of transmis- them, and spots appearing in the medium beneath and sion has not been established. around the colonies. Source: isolated from the throat of lions (Hill, 1992). Cells are short and filamentous, with occasional branch- DNA G+C content (mol%): 28 (Bd). ing. Motility for this species has not been assessed. Colo- Type strain: LL2, NCTC 11725, ATCC 49889. nies on solid medium have a typical fried-egg appearance. Sequence accession no. (16S rRNA gene): U16760. Grows well in Hayflick or SP-4 medium supplemented with 77. Mycoplasma lipofaciens Bradbury, Forrest and Williams arginine at 37°C. 1983, 334VP Opportunistic pathogen. Cause of pneumonia in domes- li.po.fa¢ci.ens. Gr. n. lipos animal fat, lard, tallow; L. v. facio tic dogs and rarely of meningitis in immunocompromised to make; N.L. part. adj. lipofaciens fat-making, intended to humans. The route of transmission is via droplet aerosol. refer to the production of a lipid film on solid media. Source: isolated from the nasopharynx, lungs, conjuncti- vae, and urogenital tract of domestic dogs and the cerebro- Cells are mainly spherical and elliptical. Nonmotile. Colo- spinal fluid of an immunocompromised human (Chalker, nies on solid medium have typical fried-egg appearance. Grows 2005). in Hayflick medium supplemented with glucose or arginine DNA G+C content (mol%): 26.7 (T ), 29.6 (Bd). at 37°C. Produces a strong “film and spots” reaction. m Type strain: PG15, ATCC 19327, NCTC 10168, NBRC Commensal of birds; little evidence exists for naturally 14848. occurring pathogenicity of the isolates, although experi- Sequence accession no. (16S rRNA gene): AF221116. mental inoculation of chicken or turkey eggs can result in embryo mortality. Inadvertent transmission to an investi- 80. Mycoplasma meleagridis Yamamoto, Bigland and Ortmayer gator during experimental inoculation studies resulted in 1965, 47AL clinical signs including rhinitis and pharyngitis. Aerosol me.le.a¢gri.dis. L. n. meleagris, -idis a turkey; L. gen. n. melea- transmission has been documented in turkeys. gridis of a turkey. Source: isolated from the infraorbital sinuses of chickens; Cells are coccoid to coccobacillary. Motility for this spe- from tissues or eggs of turkeys and ducks; and from eggs of cies has not been assessed. An extracellular capsule can be Northern goshawks (Bencˇina et al., 1987; Bradbury et al., visualized by electron microscopy following staining with 1983; Lierz et al., 2007a, b, c, 2008b). ruthenium red (Green III and Hanson, 1973). Colonies on DNA G+C content (mol%): 24.5 (Bd). solid medium are not necessarily of typical fried-egg appear- Type strain: R171, ATCC 35015, NCTC 10191. ance. Grows in Frey’s medium supplemented with arginine Sequence accession no. (16S rRNA gene): AF221115. at 37–38°C (Yamamoto et al., 1965). 78. Mycoplasma lipophilum Del Giudice, Purcell, Carski and Pathogenic; causes airsacculitis, pneumonia, sinusitis, Chanock 1974, 152AL perosis, chondrodystrophy, bursitis, synovitis, and reduced li.po.phi¢lum. Gr. n. lipos animal fat; N.L. neut. adj. philum hatchability due to embryo lethality in turkeys. Transmis- (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. sion is primarily vertical, but can also occur through droplet lipophilum fat-loving. aerosol or sexual contact. Tetracyclines, macrolides, and fluoroquinolones are Cells are pleomorphic and granular. Motility for this spe- effective chemotherapeutic agents; however, medicating cies has not been assessed. Colonies display typical fried-egg a commercial flock is not considered an effective control morphology. Growth on solid medium is associated with strategy. The temporary use of in ovo antimicrobial therapy heavy production of film that spreads over the surface of can be used to eradicate Mycoplasma meleagridis from a flock. the agar, with the development of numerous internal par- Management tactics (e.g., single age “all in/all out” systems ticles in the colonies. A film similar to that produced on and culling of endemic flocks) are more commonly utilized agar medium develops on the surface of broth-grown cul- (Kleven, 2008). Serological and molecular diagnostic meth- tures (Del Giudice et al., 1974). Grows in SP-4 or Hayflick ods have been described (Ben Abdelmoumen Mardassi medium supplemented with arginine at 37°C. et al., 2007; Ramírez et al., 2008; Raviv and Kleven, 2009). Pathogenicity for this species is unclear. This species was Source: isolated from the air sacs, trachea, infraorbital first isolated from a human patient with primary atypical sinuses, oviduct, cloaca, phallus, and eggs of turkeys, and pneumonia; however, subsequent isolations from similarly the air sacs of buzzards, kites, and kestrels (Chin et al., 2008; symptomatic patients have not been achieved. Mode of Jordan, 1979; Lam et al., 2004; Lierz et al., 2000). transmission has not been formally assessed. DNA G+C content (mol%): 27.0 (T ), 28.6 (Bd). Source: isolated from the upper and lower respiratory m Type strain: 17529, ATCC 25294, NCTC 10153. tract of a human with primary atypical pneumonia and the Sequence accession no. (16S rRNA gene): L24106. lower respiratory tract of rhesus monkeys (Hill, 1977). Further comment: previously known as avian serotype H DNA G+C content (mol%): 29.7 (Bd). (Kleckner, 1960). Type strain: MaBy, ATCC 27104, NCTC 10173, NBRC 14895. 81. Mycoplasma microti (Dillehay, Sander, Talkington, ­Thacker Sequence accession no. (16S rRNA gene): M24581. and Brown 1995) Brown, Talkington, Thacker, Brown, 604 Family I. Mycoplasmataceae

Dillehay and Tully 2001b, 412VP (Mycoplasma volis Dillehay, 84. Mycoplasma molare Røsendal 1974, 130AL Sander, Talkington, Thacker and Brown 1995, 633) mo.la¢re. L. neut. adj. molare of or belonging to a mill, here mi.cro¢ti. N.L. n. Microtus a genus of field vole; N.L. gen. n. millstone-like, referring to the heavy film reaction, which microti of Microtus. resembles the pattern on the surface of a millstone. Cells are predominantly coccoid in shape. Nonmo- Cells are coccoid to pleomorphic. Nonmotile. Colonies tile. Colonies on solid medium exhibit a typical fried-egg have a typical fried-egg appearance. Grows well in SP-4 appearance. Grows well in SP-4 supplemented with glucose medium supplemented with glucose at 37°C. A lipid film of in temperatures ranging from 35 to 37°C. characteristic appearance develops on the surface and along Opportunistic pathogen. No evidence exists for patho- the circumference of colonies grown on egg-yolk agar. genicity in the natural host; however, pneumonitis was Opportunistic pathogen. Associated with pharyngitis and experimentally induced in mice and rats (Evans-Davis et al., mild inflammatory lesions of the lower respiratory tract and 1998). may be associated with infertility, vaginitis, and posthitis of Source: isolated from the nasopharynx and lung of prairie domestic dogs. No clear evidence for primary pathogenicity voles (Dillehay et al., 1995). of the species. Mode of transmission has not been estab- DNA G+C content (mol%): not determined. lished definitively. Type strain: IL371, ATCC 700935. Source: isolated from the oral cavity, pharynx, cervix, Sequence accession no. (16S rRNA gene): AF212859. vagina, and prepuce of domestic dogs (Røsendal, 1979; (Chalker, 2005). 82. Mycoplasma moatsii Madden, Moats, London, Matthew DNA G+C content (mol%): 26.0 (Bd). AL and Sever 1974, 464 Type strain: H 542, ATCC 27746, NCTC 10144. mo.at¢si.i. N.L. gen. masc. n. moatsii of Moats, named after Sequence accession no. (16S rRNA gene): AF412985. Kenneth E. Moats, whose primary interest has been in the 85. “Mycoplasma mucosicanis” Spergser, Langer, Muck, mycoplasmas of nonhuman primates. ­Macher, Szostak, Rosengarten and Busse 2010 Cells are spheroidal and some exhibit protrusions from mu.co.si.ca¢nis. N.L. n. mucosa mucous membrane; L. n. the membrane. Motility for this species has not been canis a dog; N.L. gen. n. mucosicanis of mucous membranes assessed. Colonies exhibit typical fried-egg morphology. of a dog. Grows readily in SP-4 or Hayflick broth supplemented with either arginine or glucose at an optimum temperature of Cells are pleomorphic, but primarily coccoid. Nonmo- 37°C. tile. Colonies on solid media have a typical fried-egg mor- No evidence of pathogenicity. phology. Grows wells in modified Hayflick medium at 37°C Source: isolated from the respiratory and reproductive and produces a “film and spots” reaction. tracts of grivet monkeys and from the cecum, jejunum, and No evidence of pathogenicity. Mode of transmission has colon of wild Norway rats (Giebel et al., 1990). not been established definitively. DNA G+C content (mol%): 25.7 (Bd). Source: isolated from the prepuce, semen, vagina, cervix, Type strain: MK 405, ATCC 27625, NCTC 10158. and oral cavity of domestic dogs (Spergser et al., 2010). Sequence accession no. (16S rRNA gene): AF412984. DNA G+C content (mol%): not determined. Type strain: 1642, ATCC BAA-1895, DSM 22457. 83. Mycoplasma mobile Kirchhoff, Beyene, Fischer, Flossdorf, Sequence accession no. (16S rRNA gene): AM774638. Heitmann, Khattab, Lopatta, Rosengarten, Seidel and 86. Mycoplasma muris McGarrity, Rose, Kwiatkowski, Dion, Yousef 1987, 197VP Phillips and Tully 1983, 355VP ¢ mo bi.le. L. neut. adj. mobile motile. mu¢ris. L. n. mus, muris mouse; L. gen. n. muris of a mouse. Cells are conical or flask-shaped and have a distinct ter- Cells are primarily coccoid or coccobacillary, but exhibit minal protrusion referred to as the “head-like structure”. a few other pleomorphic forms. Motility for this species has Cells demonstrate rapid gliding motility when adhering to not been assessed. Colonies usually have a granular appear- charged surfaces and move in the directional of the head- ance and few colonies demonstrate the typical fried-egg like structure (Miyata et al., 2002, 2000). Colonies on solid appearance. Grow well in SP-4 broth supplemented with medium have a typically fried-egg appearance. Grows well arginine at 37°C and produces a “film and spots” reaction. in Aluotto’s medium supplemented with glucose or argi- No evidence of pathogenicity. nine. The temperature range for growth is 17–30°C, with Source: isolated from the vagina of a pregnant mouse (lab- optimum growth at 30°C. oratory strain RIII; McGarrity et al., 1983). Pathogenic; causes necrotic erythrodermatitis in tench. DNA G+C content (mol%): 24.9 (TLC). The mode of transmission has not been established. Type strain: RIII-4, ATCC 33757, NCTC 10196. Source: isolated from the gills of a freshwater fish Tinca( Sequence accession no. (16S rRNA gene): M23939. tinca) with “red disease” (Kirchhoff et al., 1987). DNA G+C content (mol%): 23.5 (Bd), 24.9. 87. Mycoplasma mustelae Salih, Friis, Arseculeratne, Freundt VP Type strain: 163K, ATCC 43663, NCTC 11711. and Christiansen 1983, 478 Sequence accession nos: M24480 (16S rRNA gene), mu.ste¢lae. N.L. n. Mustela (from L. n. mustela a weasel) the NC_006908 (complete genome sequence of strain generic name of the mink Mustela vison; N.L. gen. n. muste- 163K). lae of Mustela. Genus I. Mycoplasma 605

Cells are highly pleomorphic; most common morpholo- DNA G+C content (mol%): 29.2 (Bd). gies include pleomorphic rings, short filamentous forms, Type strain: MH5408, ATCC 27921, NCTC 10149. and coccoid elements. Nonmotile. Colonies on solid Sequence accession no. (16S rRNA gene): AF538961. medium show a typical fried-egg appearance. Growth in 90. Mycoplasma orale Taylor-Robinson, Canchola, Fox and Hayflick medium supplemented with glucose occurs at ­Chanock 1964, 141AL 37°C. No evidence of pathogenicity. o.ra¢le. L. n. os, oris the mouth; L. neut. suff. -ale suffix Source: isolated from the trachea and lungs of juvenile denoting pertaining to; N.L. neut. adj. orale pertaining to minks (Mustela vison; Salih et al., 1983). the mouth. DNA G+C content (mol%): 28.2 (Bd). Cells can be either coccoid or filamentous. Motility for Type strain: MX9, ATCC 35214, NCTC 10193, AMRC-C this species has not been assessed. Colonies on solid medium 1486. have a typical fried-egg appearance. Grows well in Hayflick Sequence accession no. (16S rRNA gene): AF412986. or SP-4 medium supplemented with arginine at 37°C. Mycoplasma orale is most commonly associated with con- 88. Mycoplasma neurolyticum (Sabin 1941) Freundt 1955, 73AL tamination of eukaryotic cell culture and is frequently (Musculomyces neurolyticus Sabin 1941, 57) removed by treatment of cells with antibiotics and/or main- neu.ro.ly¢ti.cum. Gr. n. neuron nerve; N.L. adj. lyticus -a -um tenance of cell lines in antibiotic-containing medium. The (from Gr. adj. lutikos -ê -on) able to loosen, able to dissolve; most effective classes of antibiotics for cell culture eradica- N.L. neut. adj. neurolyticum nerve-destroying. tion are tetracyclines, macrolides, and fluoroquinolones. Cells are filamentous and highly variable length. Nonmo- Additionally, passage of eukaryotic cells in hyperimmune tile (Nelson and Lyons, 1965). Colonies show a typical fried- serum raised against Mycoplasma orale has been shown to be egg appearance after incubation at 37°C. Grows in Hayflick an effective method of eradication (Vogelzang and Com- medium supplemented with glucose at 37°C (Naot et al., peer-Dekker, 1969). 1977). Commensal/opportunistic pathogen. Commonly found Pathogenicity is currently uncertain. Potentially associ- as a commensal of the human oral cavity; can cause respira- ated with spongiform encephalopathy and ischemic necro- tory tract infections, osteomyelitis, infectious synovitis, and sis of the brain resulting in a clinical state referred to as abscesses in immunocompromised individuals (Paessler “rolling disease” in mice and rats. Pathology may be exac- et al., 2002; Roifman et al., 1986). erbated in the presence of additional neurotropic organ- Source: isolated from the oral cavity of subclinical humans, isms (i.e., Toxoplasma gondii, Chlamydia spp., Plasmodium the sputum of an immunocompromised human with acute spp., and yellow fever virus) or during leukemic syndromes. respiratory illness, and from synovial fluid, bone, and splenic Transmission to suckling rodents occurs shortly after birth. abscesses of another immunocompromised individual.

Treatment of Mycoplasma neurolyticum infections is uncom- DNA G+C content (mol%): 24.0–28.2 (Tm, Bd). mon, as pathology is typically not resolvable after the onset Type strain: CH19299, ATCC 23714, NCTC 10112, CIP of clinical signs. 104969, NBRC 14477. Source: isolated from the brain, conjunctivae, nasophar- Sequence accession no. (16S rRNA gene): M24659. ynx, and middle ear of captive mice and rats. 91. Mycoplasma ovipneumoniae Carmichael, St George, Sullivan DNA G+C content (mol%): 22.8 (Bd), 26.2 (T ). m and Horsfall 1972, 677AL Type strain: Type A, ATCC 19988, NCTC 10166, CIP 103926, NBRC 14799. o.vi.pneu.mo.ni′ae. L. fem. n. ovis a sheep; Gr. n. pneumonia Sequence accession no. (16S rRNA gene): M23944. pneumonia; N.L. gen. n. ovipneumoniae of sheep pneumo- Further comment: a putative exotoxin with neurological nia. effects on rodents was formerly thought to be produced Morphology and motility are poorly described. The by most freshly isolated strains, although a few non-toxic organism produces a polysaccharide capsule with variable strains were described (Tully and Ruchman, 1964). The thickness that is dependent upon culture conditions and findings were not substantiated by later work (Tryon and strain (Niang et al., 1998). Colonies grown on standard Baseman, 1992). agar are convex and have a lacy or vacuolated appearance. Grows well in Friis medium or SP-4 broth supplemented 89. Mycoplasma opalescens Røsendal 1975, 469AL with glucose at 37°C. o.pa.les¢cens. L. n. opalus precious stone; N.L. neut. adj. Pathogenic; causes chronic proliferative interstitial pneu- opalescens opalescent, referring to the opalescent film pro- monia, pulmonary adenomatosis, conjunctivitis (Jones duced on solid medium. et al., 1976), and mastitis under experimental conditions Morphology by light microscopy or ultrastructural exam- (Jones, 1985) of sheep and goats. Transmission occurs via ination is not defined. Motility for this species has not been droplet aerosol and can occur via intravenous inoculation assessed. Colonies on solid medium have a typical fried-egg in experimental infection studies. appearance and possess an iridescent quality. Grows well in Source: isolated from the lungs, trachea, nose, and con- SP-4 medium supplemented with arginine at 37°C. junctivae of sheep and goats. No evidence of pathogenicity. DNA G+C content (mol%): 25.7 (Bd). Source: isolated from the oral cavity, prepuce, and pros- Type strain: Y98, NCTC 10151, ATCC 29419. tate gland of domestic dogs (Røsendal, 1975). Sequence accession no. (16S rRNA gene): U44771. 606 Family I. Mycoplasmataceae

92. Mycoplasma ovis (Neitz, Alexander and du Toit 1934) penetrans to act as a cofactor in the progression of AIDS by ­Neimark, Hoff and Ganter 2004, 369VP (Eperythrozoon ovis modulation of the immune system remains intriguing, but Neitz, Alexander and du Toit 1934, 267) in need of further substantiation (Blanchard, 1997). Trans- o¢vis. L. fem. n. ovis, -is a sheep; L. gen. n. ovis of a sheep. mission is presumed to be via sexual contact. Source: isolated from the urine of HIV-positive humans, Cells are coccoid and motility for this species has not and from the blood, respiratory secretions, and trachea of been assessed. The morphology of infected erythrocytes an HIV-negative patient with multiple autoimmune syn- is altered demonstrating a marked depression at the site dromes (Yanez et al., 1999). of Mycoplasma ovis attachment. This species has not been DNA G+C content (mol%): 30.5 (T ), 25.7 (HF-2 genome grown on artificial medium; therefore, notable biochemical m sequence; Sasaki et al., 2002). parameters are not known. Type strain: GTU-54-6A1, ATCC 55252. Neoarsphenamine is an effective therapeutic agent. Myco- Sequence accession nos: L10839 (16S rRNA gene), plasma ovis is reported to share antigens with Mycoplasma NC_004432 (HF-2 complete genome sequence). wenyonii (Kreier and Ristic, 1963), potentially complicating serology-based diagnosis of infection. 95. Mycoplasma phocicerebrale corrig. Giebel, Meier, Binder, Pathogenic; causes mild to severe anemia in sheep and Flossdorf, Poveda, Schmidt and Kirchhoff 1991, 43VP goats that often results in poor feed conversion. Transmis- pho.ci.ce.re.bra¢le. L. n. phoca seal; N.L. neut. adj. cerebrale sion occurs via blood-feeding arthropods, e.g., Haemophysalis­ of or pertaining to the brain; N.L. neut. adj. phocicerebrale plumbeum, Rhipicephalus bursa, Aedes camptorhynchus, and pertaining to the brain of a seal. Culex annulirostris (Daddow, 1980; Howard, 1975; Nikol’skii and Slipchenko, 1969), and likely via fomites such as reused Cells are coccoid or exhibit a dumbbell shape. Motility needles, shearing tools, and ear-tagging equipment (Brun- for this species has not been assessed. Colonies on solid Hansen et al., 1997; Mason and Statham, 1991). medium typically show a fried-egg appearance. Grows well Source: observed in association with erythrocytes or unat- in SP-4 medium supplemented with arginine at 37°C. tached in suspension in the blood of sheep, goats, and Pathogenic; associated with respiratory disease and con- rarely in eland and splenectomized deer. junctivitis in harbor seals (Kirchhoff et al., 1989) and a dis- DNA G+C content (mol%): not determined. tinctive ulcerative keratitis subsequent to seal bites (known Type strain: not established. as “seal finger”) and secondary arthritis in humans (Baker Sequence accession no. (16S rRNA gene): AF338268. et al., 1998; Ståby, 2004). Mode of transmission between harbor seals has not been established definitively; transmis- 93. Mycoplasma oxoniensis Hill 1991b, 24VP sion to humans appears to be zoonotic following seal bites. oxo.ni.en¢sis. N.L. adj. oxoniensis (sic) pertaining to Oxon, Source: isolated from the brains, noses, throats, lungs, and an abbreviation of Oxfordshire, where the mycoplasma was hearts of seals (Phoca vitulina) during an outbreak of respi- first isolated. ratory disease (Kirchhoff et al., 1989), and from cutaneous Cells are primarily coccoid. Nonmotile. Colonies on agar lesions of humans with seal finger (Baker et al., 1998). have a typical fried-egg appearance. Growth in SP-4 broth DNA G+C content (mol%): 25.9 (Bd). supplemented with glucose occurs at 35–37°C. Type strain: 1049, ATCC 49640, NCTC 11721. No evidence of pathogenicity. Mode of transmission is Sequence accession no. (16S rRNA gene): AF304323. unknown. Further comment: the original spelling of the specific epi- Source: isolated from the conjunctivae of the Chinese thet, phocacerebrale (sic), has been corrected by Königsson hamster (Cricetulus griseus; Hill, 1991b). et al. (2001). DNA G+C content (mol%): 29 (Bd). 96. Mycoplasma phocidae Ruhnke and Madoff 1992, 213VP Type strain: 128, NCTC 11712, ATCC 49694. pho.ci¢da.e. L. n. phoca seal; N.L. gen. n. phocidae (sic) of a Sequence accession no. (16S rRNA gene): AF412987. seal. 94. Mycoplasma penetrans Lo, Hayes, Tully, Wang, Kotani, Cells are primarily coccoid. Motility for this species has VP Pierce, Rose and Shih 1992, 363 not been assessed. Colonies on solid medium have a typi- pe.ne¢trans. L. part. adj. penetrans penetrating, referring to cal fried-egg appearance. Grows well in SP-4 or Hayflick the ability of the organism to penetrate into mammalian medium supplemented with arginine at 37°C. Produces cells. “film and spots” reaction. Cells are flask-shaped, with a distinct terminal struc- Opportunistic pathogen; associated with secondary ture reminiscent of the Mycoplasma pneumoniae attachment pneumonia of harbor seals subsequent to influenza infec- organelle. Cells demonstrate gliding motility when adher- tion. Attempts to produce disease in gray or harp seals with ing to charged surfaces and move in the direction of the Mycoplasma phocidae in pure culture were not successful terminal structure. Colonies on agar plates display a typi- (Geraci et al., 1982). Mode of transmission has not been cal fried-egg appearance. Grows well in SP-4 broth supple- established definitively. mented with either glucose or arginine at 37°C. Source: isolated from the lungs, tracheae, and heart of Opportunistic pathogen; found in the urogenital tract of harbor seals. immunocompromised humans, most notably HIV-positive DNA G+C content (mol%): 27.8 (Bd). individuals (serological detection in HIV-negative individu- Type strain: 105, ATCC 33657. als is rare). Speculation regarding the ability of Mycoplasma Sequence accession no. (16S rRNA gene): AF304325. Genus I. Mycoplasma 607

Further comment: the species designation “Mycoplasma structures. Growth is best achieved in SP-4 medium supple- ­phocae” was suggested by Königsson et al. (2001), but the mented with glucose at 37°C. original epithet “phocidae” should be retained. The sug- Pathogenic; causes interstitial pneumonitis, tracheobron- gested change is forbidden by Rule 61 (Note) of the Bacte- chitis, desquamative bronchitis, and pharyngitis [collectively riological Code because it would change the first syllable of referred to as primary atypical pneumonia (PAP); Krause the original epithet without correcting any orthographic or and Taylor-Robinson (1992)]. Less commonly, Mycoplasma typographical error. pneumoniae causes meningoencephalitis, otitis media, bullous myringitis, infectious synovitis, glomerulonephritis, pancrea- 97. Mycoplasma phocirhinis corrig. Giebel, Meier, Binder, titis, hepatitis, myocarditis, pericarditis, hemolytic anemia, Flossdorf, Poveda, Schmidt and Kirchhoff 1991, 43VP and rhabdomyolysis (Waites and Talkington, 2005). The pho.ci.rhi¢nis. L. n. phoca seal; Gr. n. rhis, rhinos nose; N.L. preceding can be primary lesions, but are often secondary gen. n. phocirhinis of the nose of a seal. to respiratory disease. Dysfunction of the immune system by Cells are coccoid; motility for this species has not been inappropriate cytokine responses or possibly molecular mim- assessed. Colonies on solid medium usually have a fried- icry following infection are associated with long-term seque- egg appearance. Grows well in Friis or Hayflick medium at lae including the development or exacerbation of asthma and 37°C. Produces “film and spots” reaction. chronic obstructive pulmonary disease; Stevens-Johnson syn- Pathogenic; associated with respiratory disease and con- drome and other exanthemas; and Guillain-Barre ­syndrome, junctivitis of harbor seals (Kirchhoff et al., 1989). Mode of Bell’s palsy, and demyelinating neuropathies (Atkinson et al., transmission has not been established definitively. 2008). Mode of transmission is via droplet aerosols (PAP) or Source: isolated from the nose, pharynx, trachea, lungs, sexual contact (urogential colonization). and heart of seals (Phoca vitulina; Kirchhoff et al., 1989). Clinical manifestations are successfully treated with tet- DNA G+C content (mol%): 26.5 (Bd). racyclines, fluoroquinolones, macrolides, and lincosamides Type strain: 852, ATCC 49639, NCTC 11722. (Waites and Talkington, 2005). Signs can be treated with Sequence accession no. (16S rRNA gene): AF304324. inhaled or injected steroids. Experimental vaccinations Further comment: the original spelling of the specific aimed at preventing infection have been unsuccessful due ­epithet, phocarhinis (sic), has been corrected by Königsson to failure to elicit immune responses, retention of viru- et al. (2001) lence, or invocation of immune responses that exacerbated clinical signs (Barile, 1984; Jacobs et al., 1988). Mycoplasma 98. Mycoplasma pirum Del Giudice, Tully, Rose and Cole 1985, pneumoniae is reported to share antigens with Mycoplasma 290VP genitalium (Taylor-Robinson, 1983a), potentially complicat- pi¢rum. L. neut. n. pirum (nominative in apposition) pear, ing serology-based diagnosis of infection. referring to the pear-shaped morphology of the cells. Source: isolated from the upper and lower respiratory Cells are predominantly flask or pear-shaped and possess tract, cerebrospinal fluid, synovial fluid, and urogential an organized terminal structure, with an outer, finely par- tract of humans.

ticulate nap covering the entire surface of the cell. Cells DNA G+C content (mol%): 38.6 (Tm), 40.0 (strain M129 exhibit low-speed gliding motility and move in the direc- genome sequence). tion of the terminal organelle (Hatchel and Balish, 2008). Type strain: FH, ATCC 15531, NCTC 10119, CIP 103766, Colonies display a typical fried-egg appearance. Grows well NBRC 14401. in SP-4 medium supplemented with glucose at 37°C. Sequence accession nos: M29061 (16S rRNA gene), U00089 No evidence of pathogenicity. (strain M129 genome sequence). Source: isolated from the rectum of immunocompetent 100. Mycoplasma primatum Del Giudice, Carski, Barile, ­Lemcke humans and whole blood and circulating lymphocytes of and Tully 1971, 442AL HIV-positive humans (Montagnier et al., 1990). Originally isolated from cultured eukaryotic cells that were of human pri.ma¢tum. L. n. primas, primatis chief, from which pri- origin (Del Giudice et al., 1985). mates, the highest order of mammals originates; L. pl. gen. DNA G+C content (mol%): 25.5 (Bd). n. primatum of chiefs, of primates. Type strain: HRC 70-159, ATCC 25960, NCTC 11702. Cells are both spherical and coccobacillary. Motility for Sequence accession no. (16S rRNA gene): M23940. this species has not been assessed. Colony morphology has a fried-egg appearance. Grows well in SP-4 or Hayflick 99. Mycoplasma pneumoniae Somerson, Taylor-Robinson and medium supplemented with arginine at 37°C. Chanock 1963, 122AL Opportunistic pathogen; rarely associated with keratitis pneu.mo.ni¢ae. Gr. n. pneumonia pneumonia; N.L. gen. n. in humans (Ruiter and Wentholt, 1955). Mode of trans- pneumoniae of pneumonia. mission has not been established. Cells are highly pleomorphic; the predominant shape Source: isolated from the oral cavity and/or urogenital includes a long, thin terminal structure at one cell pole, tract of baboons, African green monkeys, rhesus macaques, with or without a trailing filament at the opposite pole. squirrel monkeys, and humans (Hill, 1977; Somerson and Cells are motile and glide in the direction of the terminal Cole, 1979; Thomsen, 1974).

organelle when attached to cell surfaces, plastic, or glass. DNA G+C content (mol%): 28.6 (Tm). Colonies on solid medium usually lack the light peripheral Type strain: HRC292, ATCC 25948, NCTC 10163. zone, appearing rather as circular dome-shaped, granular Sequence accession no. (16S rRNA gene): AF221118. 608 Family I. Mycoplasmataceae

101. Mycoplasma pullorum Jordan, Ernø, Cottew, Hinz and Type strain: Ash, PG34, ATCC 19612, NCTC 10139, CIP Stipkovits 1982, 114VP 75.26, NBRC 14896. pul.lo¢rum. L. n. pullus a young animal, especially chicken; Sequence accession nos: M23941 (16S rRNA gene L. gen. pl. n. pullorum of young chickens. sequence), NC_002771 (strain UAB CTIP genome sequence). Cells are coccoid to coccobacillary. Motility for this 103. Mycoplasma putrefaciens Tully, Barile, Edward, Theodore species has not been assessed. Colonies on solid medium and Ernø 1974, 116AL display typical fried-egg appearance. Grows in Frey’s or pu.tre.fa¢ci.ens. L. v. putrefacio to make rotten; L. part. Hayflick medium supplemented with glucose at 37°C. adj. putrefaciens making rotten or putrefying, connot- Pathogenicity not fully established, but has been asso- ing the production of a putrid odor in broth and agar ciated with tracheitis and airsacculitis in chickens, and ­cultures. embryo lethality resulting in reduced hatchability in chickens and turkeys. Mode of transmission has not been Cells are predominantly coccobacillary to pleomorphic. assessed definitively. Nonmotile. Formation of biofilms has been demonstrated Source: isolated from the trachea, air sacs, and eggs of (McAuliffe et al., 2006). Colony morphology has a typical chickens; from the eggs of turkeys; and from tissues of fried-egg appearance. Grows well in SP-4 medium supple- pheasants, partridges, pigeons, and quail (Bencˇina et al., mented with glucose at 37°C. 1987; Bradbury et al., 2001; Kempf et al., 1991; Kleven, Pathogenic; causes polyarthritis, mastitis, conjunctivi- 2003; Lobo et al., 2004; Moalic et al., 1997; Poveda et al., tis (a syndrome collectively termed contagious agalactia) 1990). (Bergonier et al., 1997), abortion, salpingitis, metritis, and DNA G+C content (mol%): 29 (Bd). testicular atrophy (Gil et al., 2003) in goats. Type strain: CKK, ATCC 33553, NCTC 10187. Macrolides, fluoroquinolones, lincosamides, and tetra- Sequence accession no. (16S rRNA gene): U58504. cyclines are effective against Mycoplasma putrefaciens; how- Further comment: previously known as avian serotype C ever, control measures such as decontamination of fomites (Adler et al., 1958). and culling of infected herds are typically recommended to discourage the development of antimicrobial-resistant 102. Mycoplasma pulmonis (Sabin 1941) Freundt 1955, 73AL strains in carrier animals (Antunes et al., 2007; Bergonier (Murimyces pulmonis Sabin 1941, 57) et al., 1997). pul.mo¢nis. L. n. pulmo, -onis the lung; L. gen. n. pulmonis Source: isolated from the synovial fluid, udders, expelled of the lung. milk, conjunctivae, ear canal, uterus, and testes of goats. DNA G+C content (mol%): 28.9 (T ). Cells are predominantly coccoid with a well-organized m terminal structure. Cells are motile and glide in the direc- Type strain: KS1, ATCC 15718, NCTC 10155. tion of the terminal structure. An extracellular capsular Sequence accession no. (16S rRNA gene): M23938. matrix can be demonstrated by staining with ruthenium 104. Mycoplasma salivarium Edward 1955, 90AL red and formation of biofilms has been demonstrated. sa.li.va¢ri.um. L. neut. adj. salivarium slimy, saliva-like, Colonies on solid medium have a coarsely granulated and intended to denote of saliva. vacuolated appearance, with a lesser tendency to grow into the agar, and the central spot is consistently less well Cells are coccoid to coccobacillary. Nonmotile. Colonies defined than in most other Mycoplasma species. Grows in are large with a typical fried-egg appearance. Grows well in SP-4 or modified Hayflick broth supplemented with glu- Hayflick medium supplemented with arginine at 37°C and cose at an optimum temperature of 37°C. produces a “film and spots” reaction. Pathogenic; causes rhinitis, laryngotracheitis, broncho- Mycoplasma salivarium is most frequently associated pneumonia (collectively described as murine respiratory with contamination of eukaryotic cell culture and is fre- mycoplasmosis in mice), otitis media, conjunctivitis, acute quently removed by treatment of cells with antibiotics and chronic arthritis, oophoritis, salpingitis, epididymitis, and/or maintenance of cell lines in antibiotic-containing and urethritis of rodents (chiefly mice, rats, guinea pigs, medium. The most effective classes of antibiotics for cell and hamsters). Transmission occurs via aerosol, fomites, culture eradication are tetracyclines, macrolides, and flu- sexual contact, or vertically during gestation. oroquinolones. Macrolides, fluoroquinolones, and tetracyclines are Opportunistic pathogen; primarily found as a commen- effective against Mycoplasma pulmonis in vitro; however, con- sal of the human oral cavity, and rarely associated with trol measures such as decontamination of fomites, culling arthritis, submasseteric abscesses, gingivitis, and periodon- of infected colonies, and treatment of clinical signs with titis in immunocompromised patients (Grisold et al., 2008; steroids are more commonly employed in clinical settings. Lamster et al., 1997; So et al., 1983). Mode of transmission Several candidate vaccines have been described. is via direct contact with human saliva. Source: isolated from the respiratory and urogenital tracts, Source: isolated from the oral cavity, synovial fluid, den- eyes, synovial fluid, and synovial membranes of (principally tal plaque, and abscessed mandibles of humans, and the captive) rodents, and rarely from the nasopharynx of rab- nasopharynx of pigs (Erickson et al., 1988). bits and horses (Allam and Lemcke, 1975; Cassell and Hill, DNA G+C content (mol%): 27.3 (Bd). 1979; Deeb and Kenny, 1967; ­Simecka et al., 1992). Type strain: PG20, H110, ATCC 23064, NCTC 10113, DNA G+C content (mol%): 27.5–29.2 (Bd), 26.6 (strain NBRC 14478. UAB CTIP genome sequence). Sequence accession no. (16S rRNA gene): M24661. Genus I. Mycoplasma 609

105. Mycoplasma simbae Hill 1992, 520VP Cells are primarily coccoid with some irregular sim¢bae. Swahili n. simba lion; N.L. gen. n. simbae of a lion. ­flask-shaped and filamentous forms seen. Motility for this species has not been assessed. Colonies on agar usually have Cells are pleomorphic and nonmotile. Colonies on solid a fried-egg appearance when grown at 37°C. Grows well in medium have a typical fried-egg appearance. Film is pro- SP-4 medium supplemented with glucose at 34–37°C. duced by cultivation on egg yolk agar. Grows well in SP-4 Pathogenicity has not been fully established, but it is medium at 37°C. associated with conjunctivitis in the European starling, No evidence of pathogenicity. Mode of transmission has mockingbirds, blue jays, and American crows. The organ- not been established. ism is also found in clinically normal birds. Mode of trans- Source: isolated from the throats of lions (Hill, 1992). mission has not been established definitively. DNA G+C content (mol%): 37 (Bd). Source: isolated from the conjunctivae of European star- Type strain: LX, NCTC 11724, ATCC 49888. lings, mockingbirds, blue jays, American crows, ­American Sequence accession no. (16S rRNA gene): U16323. robins, blackbirds, rooks, carrion crows, and magpies 106. Mycoplasma spermatophilum Hill 1991a, 232VP (Frasca et al., 1997; Ley et al., 1998; Pennycott et al., 2005; sper.ma.to.phi¢lum. Gr. n. sperma, -atos sperm or seed; N.L. Wellehan et al., 2001). neut. adj. philum (from Gr. neut. adj. philon) friend, loving; DNA G+C content (mol%): 31 (Bd). N.L. neut. adj. spermatophilum sperm-loving. Type strain: UCMF, ATCC 51945. Sequence accession no. (16S rRNA gene): U22013. Cells are primarily coccoid. Nonmotile. Colonies are convex to fried-egg shaped and are of below-average size. 109. Mycoplasma sualvi Gourlay, Wyld and Leach 1978, 292AL Grows well in SP-4 medium supplemented with added argi- su.al¢vi. L. n. sus, suis swine; L. n. alvus bowel, womb, stom- nine under anaerobic conditions at 37°C. ach; N.L. gen. n. sualvi of the bowel of swine. Pathogenic; potentially associated with infertility, as Cells are coccobacillary and many possess organized infected spermatozoa do not fertilize ova and infected terminal structures. Nonmotile. Colonies have a typical fertilized ova were unable to implant following in vitro fried-egg appearance. Grows well in SP-4 medium supple- fertilization (Hill et al., 1987; Hill, 1991a). Mode of trans- mented with either arginine or glucose at 37°C. mission is via sexual contact. No evidence of pathogenicity. Source: isolated from the semen and cervix of humans Source: isolated from the rectum, colon, small intestines, with impaired fertility. and vagina of pigs (Gourlay et al., 1978). DNA G+C content (mol%): 32 (Bd). DNA G+C content (mol%): 23.7 (Bd). Type strain: AH159, NCTC 11720, ATCC 49695, CIP Type strain: Mayfield B, NCTC 10170, ATCC 33004. 105549. Sequence accession no. (16S rRNA gene): AF412988. Sequence accession no. (16S rRNA gene): AF221119. AL 107. Mycoplasma spumans Edward 1955, 90AL 110. Mycoplasma subdolum Lemcke and Kirchhoff 1979, 49 spu¢mans. L. part. adj. spumans foaming, presumably allud- sub.do¢lum. L. neut. adj. subdolum somewhat deceptive, ing to thick dark markings that suggest the presence of alludes to the deceptive color change that led to the globules inside the coarsely reticulated colonies. ­original erroneous description of the strains as urea- hydrolyzing. Cells are coccoid to filamentous. Motility for this species has not been assessed. Colonies in early subcultures have a Cells are coccoid to coccobacilliary. Motility for this coarsely reticulated and vacuolated appearance. A typical species has not been assessed. Colony growth on solid fried-egg appearance of the colonies develops on repeated medium exhibits the typical fried-egg appearance. Grows subculturing. Grows well in SP-4 or modified Hayflick well in SP-4, Frey’s, or Hayflick medium supplemented medium supplemented with arginine. with arginine at 37°C. Opportunistic pathogen; primarily found as a commen- Opportunistic pathogen; equivocal evidence for viru- sal of the nasopharynx, but has also been associated with lence may represent variation among strains. Associated pneumonia and arthritis of domestic dogs. Mode of trans- with impaired fecundity and abortion in horses; however, mission has not been established definitively. is highly prevalent in clinically normal horses (Spergser Source: isolated from the lungs, nasopharynx, synovial fluid, et al., 2002). Mode of transmission is via sexual contact. cerebrospinal fluid, trachea, prepuce, prostate, bladder, cer- Source: isolated from the cervix, semen, and aborted vix, vagina, and urine of domestic dogs (Chalker, 2005). foals of horses (Lemcke and Kirchhoff, 1979). DNA G+C content (mol%): 28.8 (Bd). DNA G+C content (mol%): 28.4 (Tm). Type strain: PG13, ATCC 19526, NCTC 10169, NBRC Type strain: TB, ATCC 29870, NCTC 10175. 14849. Sequence accession no. (16S rRNA gene): AF125588. Sequence accession no. (16S rRNA gene): AF125587. 111. Mycoplasma suis corrig. (Splitter 1950) Neimark, VP 108. Mycoplasma sturni Forsyth, Tully, Gorton, Hinckley, ­Johansson, Rikihisa and Tully 2002b, 683 (Eperythrozoon Frasca, van Kruiningen and Geary 1996, 719VP suis Splitter 1950, 513) stur¢ni. N.L. n. Sturnus (from L. n. sturnus a starling or stare) su¢is. L. gen. n. suis of the pig. a genus of birds, N.L. gen. n. sturni of the genus Sturnus, the Cells are coccoid. Motility for this species has not been genus of the bird from which the organism was isolated. assessed. This species has not been grown on any artificial 610 Family I. Mycoplasmataceae

medium; therefore, notable biochemical parameters are DNA G+C content (mol%): 34.2 (Bd). not known. Type strain: WVU 1853, ATCC 25204, NCTC 10124. Neoarsphenamine and tetracyclines are effective thera- Sequence accession nos: X52083 (16S rRNA gene), peutic agents. An enzyme-linked immunosorbant assay NC_007294 (strain 53 complete genome sequence). (ELISA) and PCR-based detection assays to enable diagno- 113. Mycoplasma testudineum Brown, Merritt, Jacobson, Klein, sis of infection have been described (Groebel et al., 2009; Tully and Brown 2004, 1529VP ­Gwaltney and Oberst, 1994; Hoelzle, 2008; Hsu et al., 1992). Pathogenic; causes febrile icteroanemia in pigs. Trans- tes.tu.di¢ne.um. L. neut. adj. testudineum of or pertaining mission occurs via insect vectors including Stomoxys calci- to a tortoise. trans and Aedes aegypti (Prullage et al., 1993). Cells are predominantly coccoid in shape, though some Source: observed in association with the erythrocytes of exhibit a terminal protrusion. Cells exhibit gliding motility. pigs. Colonies on solid medium exhibit typical fried-egg forms. DNA G+C content (mol%): 31.1 (complete genome Grows well in SP-4 medium supplemented with glucose at sequence of strain Illinois; J.B. Messick et al., unpublished). 22–30°C. Type strain: not established. Pathogenic; causes rhinitis and conjunctivitis in desert Sequence accession no. (16S rRNA gene): AF029394. and gopher tortoises. Mode of transmission appears to be Further comment: the original spelling of the specific epi- intranasal inhalation (Brown et al., 2004). thet, haemosuis (sic), has been corrected by the List Editor. Source: isolated from the nares of desert tortoises 112. Mycoplasma synoviae Olson, Kerr and Campbell 1964, (Gopherus agassizii) and gopher tortoises (Gopherus 209AL ­polyphemus). DNA G+C content (mol%): not determined. sy.novi¢ae. N.L. n. synovia the joint fluid; N.L. gen. n. syn- Type strain: BH29, ATCC 700618, MCCM 03231. oviae of joint fluid. Sequence accession no. (16S rRNA gene): AY366210. Cells are coccoid and pleomorphic. Nonmotile. An VP amorphous extracellular layer is described. Colony appear- 114. Mycoplasma testudinis Hill 1985, 491 ance on solid medium is variable with some showing typi- tes.tu¢di.nis. L. n. testudo, -inis tortoise; L. gen. n. testudinis cal fried-egg type colonies. Grows well in Frey’s medium of a tortoise. supplemented with glucose, l-cysteine, and nicotinamide Cells are pleomorphic, with many possessing a terminal adenine dinucleotide at 37°C. Produces a “film and spots” organelle similar to those of Mycoplasma gallisepticum and reaction (Ajufo and Whithear, 1980; Frey et al., 1968). Mycoplasma amphoriforme that periodically exhibits curva- Pathogenic; causes infectious synovitis, osteoarthritis, ture (Hatchel et al., 2006). A subset of cells are motile and and upper respiratory disease which is often subclinical in glide at high speed in the direction of the terminal struc- chickens and turkeys. Also associated with a reduction in ture. Colonies on solid medium have a typical fried-egg egg quality in chickens. Mycoplasma synoviae is often found appearance. Grows well in SP-4 medium supplemented in association with additional avian pathogens including with glucose at 25–37°C, with optimum growth at 30°C. Mycoplasma gallisepticum, avian strains of Escherichia coli, No evidence of pathogenicity. Mode of transmission has Newcastle disease virus, and infectious bronchitis virus. not been established definitively. Direct contact with fomites and droplet aerosols are the Source: isolated from the cloaca of a Greek tortoise (Hill, primary mechanisms of transmission. 1985). Tetracyclines and fluoroquinolones are effective che- DNA G+C content (mol%): 35 (Tm). motherapeutic agents; however, treatment is typically only Type strain: 01008, NCTC 11701, ATCC 43263. sought for individual birds, as medicating a commercial Sequence accession no. (16S rRNA gene): U09788. flock is not considered an effective control strategy. Vac- cination and management strategies (i.e., single age “all 115. Mycoplasma verecundum Gourlay, Leach and Howard in/all out” systems and culling of endemic flocks) are 1974, 483AL more commonly utilized. A live vaccine is commercially ve.re¢cun.dum. L. neut. adj. verecundum shy, unobtru- available. Mycoplasma synoviae shares surface antigens with sive, free from extravagance, alluding to the lack of obvi- Mycoplasma gallisepticum, potentially complicating serology- ous biochemical characteristics of the species. based diagnosis of infection. Numerous molecular diag- Cells are highly pleomorphic, exhibiting coccoid bod- nostics have been described. This organism is listed in the ies, ring forms, and branched filaments. Motility for this Terrestrial Animal Health Code of the Office International species has not been assessed. Growth on solid medium des Epizooties (http://oie.int; Yogev et al., 1989; ­Browning produces colonies with the typical fried-egg appearance. et al., 2005; Kleven, 2008; Hammond et al., 2009; Raviv and Grows well in SP-4 or Hayflick medium at 37°C. Kleven, 2009) Probable commensal; attempts to produce disease experi­ Source: isolated from the synovial fluid, synovial mem- mentally have been unsuccessful (Gourlay and Howard, branes, and respiratory tract tissues of chickens and tur- 1979). Mode of transmission has not been established. keys, and from ducks, geese, pigeons, Japanese quail, Source: isolated from the eyes of calves with conjunc- pheasants, red-legged partridges, wild turkeys, and house tivitis, the prepuce of clinically normal bulls, and from sparrows (Bradbury and Morrow, 2008; Feberwee et al., ­in-market kale (Gourlay and Howard, 1979; Gourlay et al., 2009; Jordan, 1979; Kleven, 1998). 1974; Somerson et al., 1982). Genus I. Mycoplasma 611

DNA G+C content (mol%): 27 (Tm). DNA G+C content (mol%): not determined. Type strain: 107, ATCC 27862, NCTC 10145. Type strain: not established. Sequence accession no. (16S rRNA gene): AF412989. Sequence accession no. (16S rRNA gene): AF016546. 116. Mycoplasma wenyonii (Adler and Ellenbogen 1934) 117. Mycoplasma yeatsii DaMassa, Tully, Rose, Pitcher, Leach ­Neimark, Johansson, Rikihisa and Tully 2002b, 683VP and Cottew 1994, 483VP ­( Eperythrozoon wenyonii Adler and Ellenbogen 1934, 220) ye.at¢si.i. N.L. masc. gen. n. yeatsii of Yeats, named after F.R. we.ny.o¢ni.i. N.L. masc. gen. n. wenyonii of Wenyon, named Yeats, an Australian veterinarian who was a co-isolator of after Charles Morley Wenyon (1878–1948), an investigator the organism. of these organisms. Cells are coccoid and nonmotile. Colonies on agar have Cells are coccoid. Motility for this species has not been a fried-egg appearance. Grows well in SP-4 medium sup- assessed. This species has not been grown on any artificial plemented with glucose at 37°C. Formation of biofilms has medium; therefore, notable biochemical parameters are been demonstrated (McAuliffe et al., 2006). not known. Opportunistic pathogen; commensal of the ear canal of Pathogenic; causes anemia and subsequent lameness and/ goats that has rarely been found in association with masti- or infertility in cattle. Transmission is primarily vector-mediated tis and arthritis (DaMassa et al., 1991). The mode of trans- by Dermacentor andersoni and reportedly can also occur vertically mission has not been established. during gestation. Oxytetracycline is an effective therapeutic Source: isolated from the external ear canals, retropha- agent (Montes et al., 1994). Mycoplasma wenyonii is reported to ryngeal lymph node, nasal cavity, udders, and milk of share antigens with Mycoplasma ovis (Kreier and Ristic, 1963), goats.

potentially complicating serology-based diagnosis of infection. DNA G+C content (mol%): 26.6 (Tm). Source: observed in association with the erythrocytes of Type strain: GIH, ATCC 51346, NCTC 11730, CIP cattle; Kreier and Ristic (1968) reported in addition to 105675. erythrocytes an association with platelets. Sequence accession no. (16S rRNA gene): U67946.

Species incertae sedis 1. Mycoplasma coccoides (Schilling 1928) Neimark, Peters, to be vector-borne and mediated by the rat louse Polyplex Robinson and Stewart 2005, 1389VP (Eperythrozoon coccoides spinulosa and the mouse louse Polyplex serrata. Schilling 1928, 1854) Neoarsphenamine and oxophenarsine were thought to coc.co¢ides. N.L. masc. n. coccus (from Gr. masc. n. kokkos be effective chemotherapeutic agents for treatment of Myco- grain, seed) coccus; L. suff. -oides (from Gr. suff. eides, from plasma coccoides infection in captive rodents, whereas tetracy- Gr. n. eidos that which is seen, form, shape, figure), resem- clines are effective only at keeping infection at subclinical bling, similar; N.L. neut. adj. coccoides coccus-shaped. levels (Thurston, 1953). Source: observed in association with the erythrocytes of Cells are coccoid. Motility for this species has not been wild and captive rodents. assessed. This species has not been grown on artificial medium; DNA G+C content (mol%): not determined. therefore, notable biochemical parameters are not known. Type strain: not established. Pathogenic; causes anemia in wild and captive mice, and Sequence accession no. (16S rRNA gene): AY171918. captive rats, hamsters, and rabbits. Transmission is believed

Species Candidatus 1. “Candidatus Mycoplasma haematoparvum” Sykes, Ball, 2. “Candidatus Mycoplasma haemobos” Tagawa, Matsumoto ­Bailiff and Fry 2005, 29 and Inokuma 2008, 179 ha.e.ma.to.par¢vum. Gr. neut. n. haema, -atos blood; L. neut. ha.e.mo¢bos. Gr. neut. n. haema blood; L. n. bos an ox, a bull, a adj. parvum small; N.L. neut. adj. haemoatoparvum small cow; N.L. n. haemobos (sic) intended to mean of cattle blood. (mycoplasma) from blood. Source: blood of infected cattle. Source: blood of infected canines (Sykes et al., 2005). Host habitat: blood of cattle. Host habitat: circulation of infected canines. Phylogeny: assignment to the hemoplasma cluster of the Phylogeny: assignment to the hemoplasma cluster of pneumoniae group of the genus Mycoplasma. the pneumoniae group of Mollicutes (Foley and Pedersen, Cultivation status: non-culturable. 2001). Cell morphology: wall-less; coccoid in shape. Cell morphology: wall-less; coccoid in shape. Optimum growth temperature: not applicable. Optimum growth temperature: not applicable. Sequence accession no. (16S rRNA gene): EF460765. Cultivation status: non-culturable. Further comment: this organism is synonymous with “Candidatus Sequence accession no. (16S rRNA gene): AY854037. Mycoplasma haemobovis” (sequence accession no. EF616468). 612 Family I. Mycoplasmataceae

3. “Candidatus Mycoplasma haemodidelphidis” Messick, 7. “Candidatus Mycoplasma ravipulmonis” Neimark, Mitchel- Walker, Raphael, Berent and Shi 2002, 697 more and Leach 1998, 393 ha.e.mo.di.del¢phi.dis. Gr. neut n. haema blood; N.L. fem. ra.vi.pul.mo¢nis. L. adj. ravus grayish; L. n. pulmo, -onis the gen. n. didelphidis of the opossum; N.L. gen. n. haemodidel- lung; N.L. gen. n. ravipulmonis of a gray lung. phidis of opossum blood. Source: lung tissue of mouse with respiratory infection Source: blood of an infected opossum. (gray lung disease). Host habitat: circulation of an infected opossum. Host habitat: respiratory tissue of mice with pneumonia. Phylogeny: assignment to the hemoplasma cluster of the Phylogeny: forms a single species line in the hominis group pneumoniae group of mollicutes (Messick et al., 2002). of mollicutes (Neimark et al., 1998; Pettersson et al., 2000). Cultivation status: non-culturable. Cultivation status: non-culturable. Cell morphology: wall-less; coccoid in shape. Cell morphology: wall-less; coccoid in shape; 650 nm in Optimum growth temperature: not applicable. diameter. Sequence accession no. (16S rRNA gene): AF178676. Optimum growth temperature: not applicable. Sequence accession no. (16S rRNA gene): AF001173. 4. “Candidatus Mycoplasma haemolamae” Messick, Walker, Raphael, Berent and Shi 2002, 697 8. “Candidatus Mycoplasma turicensis” Willi, Boretti, Baum- gartner, Tasker, Wenger, Cattori, Meli, Reusch, Lutz and ha.e.mo.la¢ma.e. Gr. neut n. haema blood; N.L. gen. n. lamae Hofmann-Lehmann 2006, 4430 of the alpaca; N.L. fem. gen. n. haemolamae of alpaca blood. tu.ri.cen¢sis. L. masc. (sic) adj. turicensis pertaining to Turi- Source: blood of infected llamas. cum, the Latin name of Zurich, the site of the organism’s Host habitat: circulation of infected llamas (McLaughlin initial detection. et al., 1991). Phylogeny: assignment to the hemoplasma cluster of the Source: blood of infected domestic cats. pneumoniae group of mollicutes (Messick et al., 2002). Host habitat: blood of domestic cats. Cultivation status: non-culturable. Phylogeny: assignment to the hemoplasma cluster of the Cell morphology: wall-less; coccoid in shape. pneumoniae group of the genus Mycoplasma. Optimum growth temperature: not applicable. Cultivation status: non-culturable. Sequence accession no. (16S rRNA gene): AF306346. Cell morphology: wall-less; coccoid in shape. Optimum growth temperature: not applicable. 5. “Candidatus Mycoplasma haemominutum” Foley and Sequence accession no. (16S rRNA gene): DQ157150. ­Pedersen 2001, 817 The following proposed species has been incidentally ha.e.mo.mi¢nu.tum. Gr. neut n. haema blood; L. neut. part. cited, but the putative organism remains to be established adj. minutum small in size; N.L. neut. adj. haemominutum definitively and the name has no standing in nomenclature. small (mycoplasma) from blood. Source: blood of infected felines (George et al., 2002; 1. “Candidatus Mycoplasma haemotarandirangiferis” Stoff­ Tasker et al., 2003). regen, Alt, Palmer, Olsen, Waters and Stasko 2006, 254 Host habitat: circulation of infected felines. ha.e.mo.ta.ran.di.ran.gi¢fe.ris. Gr. neut n. haema blood; Phylogeny: assignment to the hemoplasma cluster of the Rangifer tarandus scientific name of the reindeer; N.L. gen. n. pneumoniae group of mollicutes (Foley and Pedersen, haemotarandirangiferis epithet intended to indicate occur- 2001). rence in blood of reindeer. Cultivation status: non-culturable. Source: blood of reindeer. Cell morphology: wall-less; coccoid in shape; 300–600 nm in Host habitat: blood of reindeer. diameter. Phylogeny: partial 16S rRNA gene sequences suggest possi- Optimum growth temperature: not applicable. ble relationships to Mycoplasma ovis and Mycoplasma wenyonii Sequence accession no. (16S rRNA gene): U88564. (suis cluster), and/or to Mycoplasma haemofelis (haemofelis cluster). 6. “Candidatus Mycoplasma kahaneii” Neimark, Barnaud, Cultivation status: non-culturable. Gounon, Michel and Contamin 2002a, 697 Cell morphology: single punctate, chaining punctate, clus- ka.ha.ne¢i.i. N.L. masc. gen. n. kahaneii of Kahane, named tering punctate, single bacillary, chaining bacillary, single for I. Kahane. rings, chaining rings, and clustering rings. Source: blood of infected monkeys (Saimiri sciureus) Optimum growth temperature: not applicable. (Michel et al., 2000). Sequence accession nos (16S rRNA gene): DQ524812– Host habitat: circulation of infected monkeys. DQ524818. Phylogeny: assignment to the hemoplasma cluster of Further comment : sequence accession no. DQ524819, rep- the pneumoniae group of mycoplasmas (Neimark et al., resenting clone 107LSIA (Stoffregen et al., 2006), does not 2002a). support assignment of that clone to the genus Mycoplasma Cultivation status: non-culturable. because it is most similar to 16S rRNA genes of Fusobacte- Cell morphology: wall-less; coccoid in shape. rium spp. Several different partial 16S rRNA gene sequences Optimum growth temperature: not applicable. obtained from other clones are too variable to establish their Sequence accession no. (16S rRNA gene): AF338269. coherence as a species. Genus II. Ureaplasma 613

Other organisms 1. “Mycoplasma insons” May, Ortiz, Wendland, Rotstein, 3. “Mycoplasma vulturis” corrig. Oaks, Donahoe, Rurangirwa, ­Relich, Balish and Brown 2007, 298 Rideout, Gilbert and Virani 2004, 5911 in¢sons. L. neut. adj. insons guiltless, innocent. vul.tu¢ri.i. L. gen. n. vulturis of a vultures, named for the host Source: trachea and choanae of a healthy green iguana animal (Oriental white-backed vulture). (Iguana iguana). Source: lung and spleen tissue of an Oriental white-backed Host habitat: respiratory tract and blood of green iguanas vulture. (Iguana iguana). Host habitat: upper and lower respiratory tract of Oriental Phylogeny: assignment to the Mycoplasma fastidiosum cluster white-backed vulture, where it replicates intracellularly. of the pneumoniae group of the genus Mycoplasma. Phylogeny: assignment to the Mycoplasma neurolyticum clus- Cultivation status: cells are culturable in SP-4 medium sup- ter of the hominis group of the genus Mycoplasma. plemented with glucose. Cultivation status: cells can be grown in co-culture with Cell morphology: pleomorphic, but many have a highly atypi- chicken embryo fibroblasts, but have not been grown in cal shape for a mycoplasma, often resembling a twisted rod. pure in vitro culture. Optimum growth temperature: 30°C. Cell morphology: coccoid; cells display intracellular vacuoles Sequence accession no. (16S rRNA gene): DQ522159. and intracellular granules of electron-dense material. Optimum growth temperature: 37°C. 2. “Mycoplasma sphenisci” Frasca, Weber, Urquhart, Liao, Sequence accession no. (16S rRNA gene): AY191226. Gladd, Cecchini, Hudson, May, Gast, Gorton and Geary 2005, 2979 4. “Mycoplasma zalophi” Haulena, Gulland, Lawrence, Fauquier, sphe.nis¢ci. N.L. gen. n. sphenisci of Spheniscus, the genus of Jang, Aldridge, Spraker, Thomas, Brown, ­Wendland and penguin that includes the jackass penguin (Spheniscus dem- Davidson 2006, 43 ersus) from which this mycoplasma was isolated. za.lo¢phi. N.L. gen. n. zalophi of Zalophus, the genus of sea Source: choanae of a jackass penguin (Spheniscus demersus) lion that includes the California sea lion (Zalophus califor- with choanal discharge and halitosis. nianus) from which this mycoplasma was isolated. Host habitat: upper respiratory tract of the jackass pen- Source: subdermal abscesses of captive sea lions. guin. Host habitat: subdermal and intramuscular abscesses, Phylogeny: assignment to the Mycoplasma lipophilum cluster joints, lungs, and lymph nodes of captive sea lions. of the hominis group of the genus Mycoplasma. Phylogeny: assignment to the Mycoplasma hominis cluster of Cultivation status: cells are culturable in Frey’s medium the hominis group of the genus Mycoplasma. supplemented with glucose. Cultivation status: cells are culturable in SP-4 medium sup- Cell morphology: pleomorphic; some cells exhibit terminal plemented with glucose. structures. Cell morphology: not yet described. Optimum growth temperature: 37°C. Optimum growth temperature: 37°C. Sequence accession no. (16S rRNA gene): AY756171. Sequence accession no. (16S rRNA gene): AF493543.

Genus II. Ureaplasma Shepard, Lunceford, Ford, Purcell, Taylor-Robinson, Razin and Black 1974, 167AL

Ja n e t A. Ro b e r t s o n a n d Da v i d Ta y l o r -Ro b i n s o n U.re.a.plas¢ma. N.L. fem. n. urea urea; Gr. neut. n. plasma anything formed or moulded, image, figure; N.L. neut. n. Ureaplasma urea form.

Coccoid cells about 500 nm in diameter; may appear as coc- DNA G+C content (mol%): 25–32 (Bd, Tm). cobacillary forms in exponential growth phase; filaments are Type species: Ureaplasma urealyticum Shepard, Lunceford, rare. Nonmotile. Facultative anaerobes. Form exceptionally Ford, Purcell, Taylor-Robinson, Razin and Black 1974, 167 small colonies on solid media that are described either as tiny emend. Robertson, Stemke, Davis, Harasawa, Thirkell, Kong, (T) “fried-egg” colonies or as “cauliflower head” colonies hav- Shepard and Ford 2002, 593. ing a lobed periphery. Unusual pH required for growth (about 6.0–6.5). Optimal incubation temperature for examined spe- Further descriptive information cies is 35–37°C. Chemo-organotrophic. Like Mycoplasma, species Although cellular diameters as small as 100 nm and as large of Ureaplasma lack oxygen-dependent, NADH oxidase activity. as 1000 nm, and minimal reproductive units of about 330 nm Unlike Mycoplasma, species of Ureaplasma lack hexokinase or in diameter have been reported (Taylor-Robinson and Gourlay, arginine deiminase activities but have a unique and obligate 1984), published thin sections of these organisms (trivial name, requirement for urea and produce potent ureases that hydro- ureaplasmas) show diameters only as large as 450–500 nm. The lyze urea to CO2 and NH3 for energy generation and growth. exceptions are feline ureaplasmas with thin section diameters Genome sizes range from 760 to 1170 kbp (PFGE). Commensals of up to 800 nm (Harasawa et al., 1990a). Morphometric analy- or opportunistic pathogens in vertebrate hosts, primarily birds sis of cells of the type strain of Ureaplasma urealyticum fixed in and mammals (mainly primates, ungulates, and carnivores). the exponential phase of growth showed coccoid cells with 614 Family I. Mycoplasmataceae diameters of about 500 nm (Robertson et al., 1983). Similar longer incubation. Temperatures of 20–40°C are permissive for cellular diameters have been seen in hemadsorption studies in growth of examined strains, but their optimal incubation tem- which cells were pre-fixed during incubation before usual fixa- peratures are 35–37°C (Black, 1973). Ureaplasma cultures in tion for electron microscopy. Reports of budding and filamen- liquid media are incubated aerobically with growth occurring tous forms probably reflect the effect of cultural and handling in the bottom of the tube, as revealed by changes in the pH conditions on these highly plastic cells. Although the sequence indicator. Mean generation times of 10 isolates from humans of the Ureaplasma parvum genome lacks any recognizable FtsZ ranged from 50 to 105 min (Furness, 1975). Maximum titers genes (Glass et al., 2000), ureaplasmas appear to reproduce by of £108 organisms per ml of culture produce insufficient cell binary fission. Because of their minute size, ureaplasma cells mass for detectable turbidity, precluding growth measurement are rarely seen by light microscopy. Although they are of the by turbidometric or spectrophotometric methods. Growth is Gram-stain-positive lineage, the lack of cell wall results in the best measured by broth dilution methods (Ford, 1972; Rodwell organisms appearing Gram-stain-negative. They are more ­easily and Whitcomb, 1983; Stemke and Robertson, 1982). When detected if stained with crystal violet alone. Electron micro- immediate estimation of populations is required, ATP lumi- graphs have indicated hair-like structures, possibly pili, 5–8 nm nometry (Stemler et al., 1987) may be useful. An indicator sys- long, radiating from the membrane (Whitescarver and Furness, tem enhances colony detection and ureaplasma identification. 1975). An extramembranous capsule was expected from light Ammonia from urea degradation causes a rise in pH and cer- microscopic studies. In cytochemical studies, a carbohydrate- tain cations to form a golden to deep brown precipitate on the containing, capsular structure has been demonstrated in a colorless colonies, making them visible when viewed by directly strain of Ureaplasma urealyticum (Robertson and Smook, 1976; transmitted light. Initially, the urease spot test used a solution 2+ Figure 110). The structure is a lipoglycan that has also been of urea and 1 mM Mn (as MnCl2 or MnSO4) dropped onto demonstrated on the type strain T960T of Ureaplasma urealyticum agar (Shepard and Howard, 1970); later, Mn2+ was incorpo- and on a serovar 3 strain of Ureaplasma parvum; the lipoglycan rated into the agar itself to create a differential solid medium composition was strain-variable (Smith, 1986). No viruses have (e.g., ­Shepard, 1983). However, Mn2+ is toxic for ureaplasmas been seen nor has viral or plasmid nucleic acid been reported ­(Robertson and Chen, 1984). Equimolar CaCl2 (Shepard and in any ureaplasma. Robertson, 1986) gave a similar response, but allowed the recov- Ureaplasma colonies are significantly smaller in diameter ery of live cells. Manganese susceptibility has taxonomic value (£10–175 nm) than those of Mycoplasma species (300–800 nm; (Table 138); animal isolates show differing responses (Stemke Figure 110). For this reason, they were first described as “tiny et al., 1984; Stemler et al., 1987). (T) form PPLO (pleuropneumonia-like organisms) colonies” Ureaplasma urealyticum has some elements of the glyco- (Shepard, 1954) and later called T-mycoplasmas (Meloni et al., lytic cycle and pentose shunt (Cocks et al., 1985), but can- 1980; Shepard et al., 1974). Cultures on solid media may grow not degrade glucose and lacks arginine deiminase (Woodson in air, but more numerous and larger colonies result in 5–15% et al., 1965). Instead, ureaplasmas have a unique and absolute

CO2 in N2 or H2 (Robertson, 1982). Colonies of many strains requirement for urea (0.4–1.0 mM) and a slightly acidic envi- are detectable after overnight incubation and reach maximum ronment (pH 6.0–7.0); pH values outside this range can be dimensions within 2 d. Isolates from ungulates may require associated with growth inhibition. The essential ­cytoplasmic

Figure 110. Ureaplasma colonial size and cellular morphology. (a) Many isolated Ureaplasma urealyticum colonies, accentuated by a urease spot test, surround a single large Mycoplasma hominis colony on a solid genital mycoplasma (GM) agar surface. Colonies of Ureaplasma urealyticum commonly have diameters of 15–125 mm; the diameter of the Mycoplasma hominis colony shown is approximately 0.9 mm. (Reproduced with permission from Robertson et al., 1983. Sexually Transmitted Diseases 10 (October-December Suppl.): 232–239 © Lippincott Williams & Wilkins.) (b) Transmission electron micrograph of Ureaplasma urealyticum, serovar 4, strain 381/74 cells, showing coccoid morphology, extramembranous capsule stained with ruthenium red, lack of a cell wall, the single limiting mem- brane, and apparently simple cytoplasmic contents in which only ribosomes are clearly evident. Cell diameters were 485–585 nm. (Reproduced with permission from Robertson and Smook, 1976. Journal of Bacteriology 128: 658–660.) Genus II. Ureaplasma 615

Table 138. Phenotypic characteristics that partition the human serovar-standard strains of ureaplasmas to Ureaplasma speciesa

Characteristic U. urealyticum serovar U. parvum serovar Reference Isoelectric focusing and SDS-PAGE at pH 5.3 polypeptide Sayed and Kenny (1980) patterns: Absent 1, 3T, 6 Present 2, 4, 5, 7, 8T 1-D SDS-PAGE T960T biovar band:b Howard et al. (1981) Absent 1, 3T, 6 Present 2, 4, 5, 7, 8T 2-D SDS-PAGE:b Biovar 1 pattern 1, 3T, 6, 14 Mouches et al. (1981) Biovar 2 pattern 2, 4, 5, 7, 8T, 9, 11, 12 Swensen et al. (1983) Growth inhibition by 1 mM Mn2+: Robertson and Chen (1984) Temporary 1, 3T, 6, 14 Permanent 2, 4, 5, 7, 8T, 9–12c Polypeptide recognized by immunoblots: 51 and 58 kDa 1, 3T, 6, 14 Horowitz et al. (1986) 47 kDa 2, 4, 5, 7, 8T, 9–13 Biovar 1 pattern 1, 3T Lee and Kenny (1987) Biovar 2 pattern 2, 4, 8T mAb UU8/39 recognition of membrane proteins: Thirkell et al. (1989) 17 kDa only 1, 3T, 6, 14 16/17 kDa 2, 4, 5, 7, 8T, 9–13 mAb UU8/17 recognition of 72 kDa urease subunit:d Thirkell et al. (1990) No 1, 3T, 6, 14 Yes 8T mAb VB10 recognition of 72 kDa urease subunit: MacKenzie et al. (1996) Yes 1, 3T, 6, 14 No 2, 4, 5, 7, 8T, 9–13 Urease dimorphism: Davis et al. (1987) Lower MW 1, 3T, 6, 14 Higher MW 2, 4, 5, 7, 8T, 9–13 Pyrophosphatase dimorphism: Davis and Villanueva (1990) Lower MW 1, 3T, 6, 14 Higher MW 2, 4, 5, 7, 8T, 9–13 Diaphorase bands dimorphism: Davis and Villanueva (1990) Inapparent 1, 3T, 6, 14 Apparent 2, 4, 5, 7, 8T, 9–13 aT, Type strain of species. b1-D, One-dimensional; 2-D, two-dimensional. cSerovar 13 gave an intermediate response and was excluded from the initial partition scheme. dHyperimmune rabbit sera and acute and convalescent sera from women with postpartum fever. urease, comprising three subunits (Blanchard, 1990), pro- on additional physiological traits, see: Black (1973); Shepard duces a transmembrane potential, which leads to ATP syn- et al., (1974); Shepard and Masover (1979); Taylor-Robinson thesis (Romano et al., 1980; Smith et al., 1993). Ureaplasma and ­Gourlay (1984); and Pollack (1986). dependence upon catalysis of urea for energy is the basis Except for the obligatory requirements for supplementary of growth inhibition by the urease inhibitor hydroxamic urea and lower pH, ureaplasma growth requirements are simi- acid and its derivatives (Ford, 1972) and by fluorofamide lar to those of members of the genus Mycoplasma. The sterol (Kenny, 1983). The proton pump inhibitor lansoprazole and requirement is met by horse, bovine, or fetal bovine serum. its metabolites interfere with ATP synthesis at micromolar Because heat-sensitive pantothenic acid is a growth factor sup- concentrations (Nagata et al., 1995). Their small genomes plied by serum (Shepard and Lunceford, cited by Shepard and make ureaplasmas dependent upon the host for amino acids, Masover, 1979), the serum supplement should not be “inacti- amino acid precursors, lipids, and other growth components. vated” by heating to reduce complement activity. The effect of Ureaplasmas from humans exhibit minimal levels of acetate yeast extract is variable, perhaps depending upon the particular kinase activity (Muhlrad et al., 1981). Like other Mycoplasmata- strain requirements or the batch of extract. Other defined addi- ceae, ureaplasmas make superoxide dismutase (O’Brien et al., tives have been reported to enhance growth but, in the absence 1983), but, unlike the rest of this family, oxygen-dependent of a defined medium, evaluation is difficult. Dependence on NADH oxidase has not been detected (Masover et al., 1977). sterols for the integrity of the cell membrane renders ureaplas- The Ureaplasma parvum genome sequence includes genes for mas susceptible to digitonin and certain antifungal agents, but six hemin and/or Fe3+ transporters which are believed to be most strains tolerate the polyene nystatin (50 U/ml) that pre- related to respiration (Glass et al., 2000). For information vents overgrowth by yeasts. 616 Family I. Mycoplasmataceae

Information about ureaplasma genetics is now abundant. The in urine in vitro and produce urinary calculi in animal models genomes of 26 strains of Ureaplasma, including six of the seven (Reyes et al., 2009). They are found in patients with infection named species and six unnamed strains, range in size from 760 stones more often than in those with metabolic stones ­(Grenabo to 1170 kbp (Kakulphimp et al., 1991; Robertson et al., 1990). et al., 1988). A statistical association with infection stones has The largest genome belongs to Ureaplasma felinum. The G+C been made (Kaya et al., 2003). content of ureaplasmal DNA is in the range 25.5–31.6 mol%, Evidence for the association of ureaplasmas with acute which is lower than that for other Mollicutes and for all other nongonococcal urethritis in men has been controversial, but prokaryotes, greatly limiting the degeneracy in the genetic code a significant association of Ureaplasma urealyticum (but not Ure- of ureaplasmas. For the type strain of Ureaplasma urealyticum, aplasma parvum) with this disease in two of three recent studies UGA is a codon for tryptophan (Blanchard, 1990). The entire suggests a way forward to resolving this issue. Evidence for a sequences of the genomes of two strains of Ureaplasma parvum role for ureaplasmas in acute epididymitis is, at the most, mea- serovar 3 (formerly known as Ureaplasma urealyticum biovar 1; ger and it is very unlikely that they have a role in chronic prosta- Glass et al., 2000) have been determined. At 752 kbp, the organ- titis. Ureaplasmas have been associated with bacterial vaginosis isms share with other obligate symbions, such as Mycoplasma and pelvic inflammatory disease, but are unlikely to be causal in genitalium (580 kbp) and Buchnera aphidicola (641 kbp), greatly either condition. It follows, therefore, that there is no convinc- reduced genomes. Of 641–653 total genes, 32–39 code for struc- ing evidence to implicate ureaplasmas as an important cause tural RNAs and about 610 code for proteins, about 47% of which of infertility in men or in couples. The most convincing data have been classified as hypothetical genes of unknown function. relating ureaplasmas to poor pregnancy outcomes have been Some (19%) resemble genes present in other genomes, but many seen when the organisms have been detected in amniotic fluid (28%) appear unique. Unexpectedly absent in the Ureaplasma prior to membrane rupture, but there are conflicting opinions parvum genome are recognizable genes for FtsZ, for the GroEl about the role of Ureaplasma urealyticum vs Ureaplasma parvum. and GroES chaperones, and for ribonucleoside-diphosphate Data since the 1980s have supported the association of neonatal reductase. Attempts have been made to reconcile the anomalies ureaplasma infection with chronic lung disease and sometimes of gene functions assigned to this serovar of Ureaplasma parvum death in very low birth-weight infants. The ability of Ureaplasma with the activities and pathways found in various ureaplasmas parvum to induce chorioamnionitis and to contribute to pre- of humans (Glass et al., 2000; Pollack, 2001). One limitation to term labor and fetal lung injury is supported by experimental such analysis is that most of the physiological data that has been studies in rhesus monkeys (Novy et al., 2009). accumulated pertain not to Ureaplasma parvum but instead to Information on the range of animal species infected with the type species, Ureaplasma urealyticum strain T960T, possess- ureaplasmas and their geographic distribution is patchy, possi- ing a 7% larger genome. The entire sequences of the genomes bly because ureaplasmas are largely avirulent or, at least, not an of Ureaplasma urealyticum serovars 8 and 10 (Glass et al., 2000, economic threat. Isolations have been reported from squirrel, 2008) have also been determined and the genomes of the type talapoin, patas, macaque and green monkeys; as well as mar- strains of all remaining Ureaplasma urealyticum and Ureaplasma mosets and chimpanzees (Taylor-Robison and Gourlay, 1984). parvum serovars are nearly completely annotated (Glass et al., Also, there are reports of isolation from domestic dogs, raccoon 2008). Preliminary comparisons found that in addition to “core” dogs, cats and mink; cattle, sheep, goats, and camels; chickens and “dispensable” genomes for each species, Ureaplasma urealyti- and other fowl; and swine (the latter needing confirmation). cum had partly duplicated multiple-banded antigen (mba) genes Baboons, rats and mice are susceptible to experimental infec- (Kong et al., 1999a, 2000), and up to twice the number of genes tion with ureaplasmas. While Ureaplasma diversum can inhabit all that Ureaplasma parvum has for lipoproteins. Such comparisons mucosal membranes of cattle, the two natural diseases it causes are expected to substantially improve understanding of ure- are subclinical respiratory infections in young calves, which aplasmal pathogenicity and differential strain virulence. occasionally develop into bronchopneumonia, and the eco- Clinical studies based entirely on qualitative assessments of nomically important urogenital infections transmitted by bulls ureaplasmas are often difficult to interpret and only a few inves- or their semen. The latter present as vulvovaginitis or ascend tigators have presented quantitative data (Bowie et al., 1977; to cause infertility or abortion (Ruhnke et al., 1984; ter Laak De Francesco et al., 2009; Heggie et al., 2001; Taylor-Robinson et al., 1993). A species-specific PCR assay (Vasconcellos Cardosa et al., 1977). Factors such as hormonal levels, specific genetic et al., 2000) can circumvent culture insensitivity. As in humans, attributes, and even socio-economic conditions may encour- ureaplasmal diseases in animals may be influenced by the par- age urogenital colonization and proliferation. Nevertheless, ticular ureaplasma strain or many other factors. Although ure- it is clear that ureaplasmas are commensals that, on occasion, aplasmas may be present initially in certain organ and primary contribute to disease in susceptible human hosts. Infections cell cultures, the lack of urea and higher pH in most eukaryotic attributed to ureaplasmas are often associated with an immu- cell culture systems would discourage persistence. nological component (Bowie et al., 1977), including the subset Although the spectrum of diseases of primary ureaplasmal of the human population with common variable hypogam- etiology remains controversial, many potential virulence factors maglobulinemia (Cordtz and Jensen, 2006; Furr et al., 1994; have been identified. Structural elements include a capsule, Lehmer et al., 1991; Webster et al., 1978), in which ureaplasma- pilus-like fibrils, and the antigens of the outer membrane that induced septic arthritis and occasionally persistent ureaplasmal constitute the serovar determinants. Erythrocytes from several urethritis is seen (Taylor-Robinson, 1985). Sexually acquired, animal species adhere firmly to colonies of certain strains of reactive arthritis is usually linked to Chlamydia trachomatis, but Ureaplasma parvum (Shepard and Masover, 1979), but most immunological evidence of ureaplasmal involvement exists human isolates exhibit transient or no binding (Robertson ­(Horowitz et al., 1994). Through urea metabolism, ureaplasmas and Sherburne, 1991). Strains of both Ureaplasma urealyticum can induce crystallization of struvite and calcium phosphates and Ureaplasma parvum adhere to HeLa cells (Manchee and Genus II. Ureaplasma 617

Taylor-Robinson, 1969) and to spermatozoa (Knox et al., sides, chloramphenicol, and newer fluoroquinolones inhibit 2003). Demonstration of beta-hemolysis of erythrocytes by ure- ureaplasmas, but are inappropriate for broad clinical use. Fluo- aplasmal products depends upon several variables; hemolysis roquinolone resistance is increasing (Xie and Zhang, 2006) and of guinea pig erythrocytes has been most consistently observed has been found in a previously susceptible strain (Duffy et al., (Black, 1973; Manchee and Taylor-Robinson, 1970; Shepard, 2006). Ureaplasma strains exhibiting resistance to multiple anti- 1967). Manchee and Taylor-Robinson (1970) described hemo- biotics have been found in immunocompromised hosts. In one lysis of homologous erythrocytes by a canine ureaplasma as per- case, the isolates were of the same serovar, but exhibited differ- oxide-associated and blocked lysis by adding catalase, except in ent susceptibility patterns at different anatomical sites (Lehmer the presence of a catalase inhibitor. Genome sequencing has et al., 1991). Because of ongoing changes in antimicrobial sus- revealed genes resembling those for the hemolysins HlyA and ceptibility patterns, the recent literature should be consulted HlyC of enterohemorrhagic Escherichia coli (Glass et al., 2000). (e.g., Beeton et al., 2009). To treat natural Ureaplasma diversum Ammonia and ammonium ions generated by urea hydrolysis infections, tiamulin hydrogen fumarate, a diterpene agent in and the alkaline environment that they create are inhibitory common use in veterinary medicine, may be at least as effective to the ureaplasmas (Ford and MacDonald, 1967; Shepard and as the macrolide tylosin (Stipkovits et al., 1984). Others have Lunceford, 1967). They have well-established deleterious effects examined the efficacy of single or combinations of antibiotics on eukaryotic cell and mammalian tissue cultures and may be in eradicating ureaplasmas from various sites in cattle as well the “toxin” described but never substantiated (Furness, 1973). as from semen used for artificial insemination (ter Laak et al., An IgA1 protease activity has been demonstrated by ureaplas- 1993). The urease inhibitor, fluorofamide, has been used with mas from both humans and canines. Human IgA1-specific pro- varying success in eliminating ureaplasmas from animals. tease activity, apparently similar to the type 2 serine protease of When establishing antibiograms for ureaplasmas, the require- certain bacterial pathogens of humans (Spooner et al., 1992), is ments established for common bacterial pathogens do not suf- produced by both Ureaplasma parvum and Ureaplasma urealyticum fice. First, medium components may affect antibiotic activity. (Kilian et al., 1984; Robertson et al., 1984), but a gene responsi- For instance, serum-binding reduces tetracycline activity, while ble for the activity has not yet been identified. Putative phopho- the initial acidic culture reduces macrolide activity against ure- lipase A1, A2, and C activities (De Silva and Quinn, 1986) could aplasmas (Robertson et al., 1981). Conventional bacteria of not be confirmed, nor could such gene sequences be identified established, low-level susceptibility can be used to measure the (J. Glass, unpublished). Biofilm production has been described effect of the ureaplasmal medium on a particular antimicro- recently (García-Castillo et al., 2008). Ureaplasmas have been bial agent. Second, the relatively slow growth of ureaplasmas seen intracellularly during studies in cell cultures (Mazzali as compared with many pathogenic bacteria requires that the and Taylor-Robinson, 1971), but may be there transiently after half-life of the antibiotic be considered when test inoculum and phagocytosis. Cells in culture (Li et al., 2000), in either experi- incubation period are established. Lastly, the end points of the mental infection models (Moss et al., 2008) or epidemiological sensitivity tests on agar are about four-fold lower than for tests study subjects (Buss et al., 2003; Dammann et al., 2003; Jacobs- in broth (Waites et al., 1991). On consideration of the in vivo son et al., 2003; Shobokshi and Shaarawy, 2002), have exhibited environment in which these organisms naturally occur, inter- proinflammatory responses. While assessment of clinical studies pretation of susceptibility test end points continues to present is sometimes difficult because of inherent reporting bias (Klas- a challenge. An international subcommittee under the aegis sen et al., 2002; Schelonka et al., 2005), it is anticipated that of the National Committee for Clinical Laboratory Standards the application of bioarray technologies will lead to improved Institute (USA) is currently finalizing a “Final Report for Devel- understanding of ureaplasmal mechanisms of pathogenesis. opment of Quality Control Reference Standards and Methods Reviews of in vitro methodologies used for antimicrobial for Antimicrobial Susceptibility Testing” for Ureaplasma and susceptibility testing for both human (Bébéar and Robertson, Mycoplasma species infecting humans that have demonstrated 1996; Waites et al., 2001) and animal (Hannan, 2000) isolates variability in response to antimicrobial agents. are available. Antimicrobial susceptibility patterns of Ureaplasma urealyticum and Ureaplasma parvum appear to be similar (Matlow Enrichment and isolation procedures et al., 1998). The choice of therapeutic agents active against Media formulations in current use for the cultivation of them is limited. Tetracyclines or the macrolides (excluding lin- ­ureaplasmas from human sources include, in order of decreasing comycin and clindamycin) are the bacteriostatic agents usually supplementation: the 10B broth of Shepard and Lunceford employed. Ureaplasmas from human, simian, bovine, caprine, (Shepard, 1983); Taylor-Robinson’s broth (Taylor-Robinson, feline, and avian sources (Koshimizu et al., 1983) withstand 1983a), made without thallium acetate; and bromothymol blue relatively high levels of lincomycin (10 mg/ml), to which most broth (Robertson, 1978). Ureaplasmas are exquisitely sensitive to mycoplasmas are susceptible. Ureaplasmas also show in vitro thallium acetate and it should not be used to inhibit other bacte- resistance to rifampin and sulfonamides. Clinical resistance of ria. The U4 formulation for ungulate isolates was developed by ureaplasmas to tetracyclines has been long known (Ford and Howard et al. (1978). Consult Waites et al. (1991) for additional Smith, 1974). This high-level resistance is determined by the media formulations for isolation from humans, and ­Shepard presence of the tetM determinant (Roberts and Kenny, 1986), (1983), Livingston and Gauer (1974), or Hannan (2000) for iso- which is now readily identified by PCR (Blanchard et al., 1997). lation from animals. The quality of medium components may be Tetracycline-resistant strains exposed to a variety of antibiot- as important as the medium formulation. Serum supplements ics demonstrate a broad range of responses (Robertson et al., should be tested for their ability to support growth of the ure- 1988). Clinical resistance to macrolides has also been reported aplasmas of interest. Liquid medium may be stored at −20°C until (e.g., Taylor-Robinson and Furr, 1986). Certain aminoglyco- required. ­Transport medium (e.g., 2SP) should be free of antibi- 618 Family I. Mycoplasmataceae otics inhibitory to ureaplasmas. Broth cultures should be diluted ­Robertson et al., 1994) and the 16S–23S rRNA intergenic spacer to ³1:100 to reduce effects of any growth inhibitors present in the regions of all named species plus the 14 serovars associated with specimen. In general, Ureaplasma parvum is less demanding nutri- humans (Ureaplasma urealyticum and Ureaplasma parvum) have tionally than Ureaplasma urealyticum and isolates from human and been determined (Harasawa and Kanamoto, 1999; Kong et al., other animal sources together are less demanding than those 1999b). The genus Ureaplasma comprises two subclusters within from ungulate hosts. Agar surfaces are examined at ³40× magni- the highly diverse pneumoniae group of the family Mycoplasma- fication. The less fastidious strains (e.g., Ureaplasma parvum type taceae (Johansson, 2002). One subcluster contains the human, strain) are more likely to produce recognizable “fried-egg” colo- avian, and mink isolates and the other contains feline, canine, nies than are the more fastidious strains (e.g., Ureaplasma urealyti- and bovine strains (see genus Mycoplasma Figure 109, pneumo- cum type strain), which are more likely to produce “cauliflower nia group) Although the three serovars A, B, and C of Ureaplasma head” or core colonies. Cultures may be thrice cloned and the diversum are antigenically heterogeneous, the strains examined resultant culture used to initiate stocks. meet the 70% DNA–DNA hybridization benchmark used as an arbitrary species criterion. However, the available DNA–DNA Maintenance procedures hybridization values suggest that Ureaplasma gallorale and Ure- Broth cultures of ureaplasmas commonly become sterile within aplasma canigenitalium might each represent more than a single 12–24 h incubation at 35–37°C. “Red is dead” was the mantra of species. The 16S rRNA gene sequences of ureaplasmas isolated Shepard in regard to his phenol red-containing broth medium. from nonhuman primates and some less-studied vertebrates are One way to lessen this problem is to change to an indicator that unknown. The range of G+C contents of ureaplasmal DNA is changes color at a lower pH (Robertson, 1978). To Shepard’s too narrow to have much taxonomic utility. Nevertheless, the mantra we might add, “the higher the urea concentration, the values for isolates from cattle, sheep, and goats are between steeper the death phase”, an effect not countered by buffer. Incu- 28.7 and 31.6 mol% (Howard et al., 1978), at the higher end of bation of diluted cultures at 30–34°C slows growth and main- the range for the genus. tains viability for up to 1 week, reducing the frequency of culture It is generally assumed that all ureaplasmas from avian transfer and helping in transport. Short exposure of broth cul- sources belong to the species Ureaplasma gallorale. The seven tures to refrigeration reduces viability. Cells within colonies on avian isolates examined were similar to each other serologically solid medium may be recovered for approximately 1 week if the and in SDS-PAGE, immunoblot, and RFLP profiles, and they cultures are removed from incubation when growth is detect- were distinct from Ureaplasma urealyticum and Ureaplasma diver- able and stored under cool, humidified conditions. For long- sum, although resemblances between Ureaplasma gallorale and term storage, ultra-low temperatures (−80°C or liquid nitrogen) Ureaplasma urealyticum based upon one- and two-dimensional with a cryoprotectant [e.g., 10–20% (v/v) sterile glycerol] can PAGE analyses have been reported (Mouches et al., 1981). How- maintain viability for well over a decade. For lyophilization, the ever, the DNA–DNA hybridization values among Ureaplasma gal- pellets from broth cultures centrifuged at high speed are resus- lorale strains fall into two clusters. Homology was 70–100% for pended in a minimal volume of liquid medium or the serum the five strains within cluster A and 96–100% for the two strains used for its supplementation before processing; on reconstitu- within cluster B that were studied. Between strains of cluster tion, urease activity occasionally is not immediately evident. A and B, homology was only 51–59% and, vice versa, 52–69% (Harasawa et al., 1985). The taxonomic status of avian ureaplas- Differentiation of the genus Ureaplasma from other genera mas, therefore, might benefit from examination of additional Properties that partially fulfill criteria for assignment to the strains and reconsideration. The clusters appear to be more class Mollicutes (Brown et al., 2007) include absence of a cell closely related than Ureaplasma urealyticum and Ureaplasma par- wall, filterability, and the presence of conserved 16S rRNA vum (Table 139). gene sequences. Aerobic or facultatively anaerobic growth in The taxonomy of ureaplasmas of canines is also problematic. artificial media and the necessity for sterols for growth exclude The first report indicated G+C contents of 27.2–27.8 mol% assignment to the genera Anaeroplasma, Asteroleplasma, Achole- (Bd; Howard et al., 1978). Later, the representatives of four plasma, and “Candidatus Phytoplasma”. Non-spiral cellular mor- serogroups (SI to SIV for strains DIM-C, D29M, D11N-A, and phology and regular association with a vertebrate host or fluids D6P-CT, respectively) were reported to have G+C contents of of vertebrate origin support exclusion from the genera Spiro- 28.3–29.4 mol% (HPLC). However, the published data regard- plasma, Entomoplasma, and Mesoplasma. The ability to hydrolyze ing their DNA reassociation values are confusing. Initially, urea, with the inability to metabolize either glucose or arginine, Barile (1986) and Harasawa et al. (1990b) reported that the excludes assignment to the genus Mycoplasma. For routine pur- serogroup SI representative, DIM-C, had 73% homology with poses, the colonial morphology characteristic of ureaplasmas the serotype 2 SII representative, D29M, indicating that these and demonstration of the isolate’s ability to catabolize urea, two strains likely belong to the same species. However, perhaps using the urease spot test or indicator agar, suffice for prelimi- because of the borderline value, further work was undertaken, nary differentiation from other mollicutes. Other methods for also using [3H]DNA–DNA hybridization procedures (Harasawa urease detection have been largely replaced by PCRs that detect et al., 1993). The values obtained ranged from 41 to 63% homol- urease genes. ogy among the four serogroups, i.e., each serogroup seemed to represent a distinct species. At present, the only named canine Taxonomic comments ureaplasma species is Ureaplasma canigenitalium; the SIV rep- The hypothetical evolutionary relationships of the Mollicutes resentative D6P-CT is the type strain (Harasawa et al., 1993). have been based primarily upon 16S rRNA gene sequences Its degree of distinctiveness from the other serogroups is not (Weisburg et al., 1989). The nucleotide sequence for the emphasized in the literature. 16S rRNA genes (Kong et al., 1999b; Robertson et al., 1993; Table 139. Genotypic characteristics that partition the serovar-standard strains of ureaplasmas isolated from humans to the level of speciesa

Characteristic U. urealyticum serovars U. parvum serovars Reference(s) DNA–DNA relatedness with strain 27T DNA: Christiansen et al. (1981) 91–102% 1, 3T, 6 38–60% 2, 4, 5, 7, 8T DNA–DNA relatedness with strain T960T DNA: Christiansen et al. (1981) 49–52% 1, 3T, 6 69–100% 2, 4, 5, 7, 8T DNA–DNA relatedness with strain 27T DNA: Harasawa et al. (1991) 75–100% 1, 3T, 6, 14 38–57% 2, 4, 5, 7, 8T, 9–13 DNA–DNA relatedness with strain T960T DNA: Harasawa et al. (1991) 48–59% 1, 3T, 6, 14 76–101% 2, 4, 5, 7, 8T, 9–13 Cleavage of DNA by Fnu4HI: Cocks and Finch (1987) Yes 1, 3T, 6 No 2, 4, 5, 7, 8T, 9 BamHI, HindIII, and PstI RFLP probed with RNA genes: Razin (1983) Biovar l pattern 1, 3T, 6 Biovar 2 pattern 2, 4, 5, 7, 8T, 9 EcoRI and HindIII RFLP probed with RNA genes: Harasawa et al. (1991) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13b Genome sizes determined by PFGE: Robertson et al. (1990) ~760 kbp 1, 3T, 6, 14 840–1140 kbp 2, 4, 5, 7, 8T, 9–13 Heterogeneity of alpha polypeptide-associated urease genes: Blanchard (1990) Yes 1, 3T, 6, 10c, 12c, 14 No 2, 4, 5, 7, 8T, 9, 13 Heterogeneity of HindIII site in subunit ureC of urease gene: Neyrolles et al. (1996) Absent 1, 3T, 6 Present 2, 8T Heterogeneity of HindIII fragments probed with Neyrolles et al. (1996) serovar 8IC61 urease probe: Biovar 1 pattern 1, 3T, 6 Biovar 2 pattern 2, 8T Heterogeneity of urease subunit-associated genes:d Kong et al. (1999b) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13 Heterogeneity of biovar-specific 16S rRNA genes determined Robertson et al. (1993) by PCR:d Strain 27T 1, 3T, 6, 14 Strain T960T 2, 4, 5, 7, 8T, 9–13 Biovar-specific 16S rRNA gene sequences:d Robertson et al. (1994) Strain 27T sequence 1, 3T, 6, 14 Strain T960T sequence 2, 5, 8T 16S rRNA, spacer regions, and urease subunit sequences:d Kong et al. (1999b) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13 RFLP of 5¢ region of mba genes: Teng et al. (1994) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13 5¢ end sequences of mba genes:d,e Kong et al. (1999b) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13 16S–23S intergenic spacer region:d Harasawa and Kanamoto (1999), Kong et al. (1999b) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13 Arbitrarily primed PCR:d Grattard et al. (1995) Biovar 1 pattern 1, 3T, 6, 14 Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

aT, Type strain of species. bSerovar 13 response to EcoRI was anomalous. cThis anomalous pattern resulted from cultures being misidentified as serovars 10 and 12; the error was corrected by Teng et al. (1994). Kong et al. (1999b) have con- firmed the efficacy of the Blanchard (1990) primers. dPrimers used for biovar-defining PCR(s). eThe multiple band (MB) antigens seen by PAGE are putative virulence markers. See text for details. 620 Family I. Mycoplasmataceae

While DNA–DNA hybridization has been key to species level period representing antigens in Vancouver, BC, Canada (Ford, identification for the genus Ureaplasma, serology led to our knowl- 1967), Camp Lejeune, NC (Shepard, 1954), and Boston, MA, edge of intra-species relationships (Robertson et al., 2002) and USA (Lin et al., 1972), i.e., on the west and east coasts of North still figures importantly. Much interest has been focused specifi- America. While putative untypable strains are occasionally cally on the multiple-banded antigens (MBA) of the ureaplas- encountered, after cloning, these have usually turned out to be mal cell surface. Watson et al. (1990), studying the type strain serovar 3, probably the most likely to dominate in a mixed cul- (serovar 3) of Ureaplasma parvum, identified the predominant ture. However, additional serovars/genovars can be expected to ureaplasmal antigens recognized by the host during human emerge, especially from other parts of the world. infection. The MBA were first seen as unusual, laddered bands The named species were identified primarily by DNA–DNA in immunoblots. These lipoproteins exhibited epitopes for both hybridization and by serological tests. For taxonomic studies, serovar specificity and cross-reactivity, and showed in vitro size the metabolism inhibition test (Purcell et al., 1966; Robertson variation (Teng et al., 1994; Zheng et al., 1995). Monecke et al. and Stemke, 1979; Taylor-Robinson, 1983b) and either a direct (2003) added evidence that the MBA are involved in a phase- or indirect immunofluorescence test (Black and Krogsgaard- switching process similar to that identified in several Mycoplasma Jensen, 1974; Piot, 1977; Stemke and Robertson, 1981) have species. The mba gene sequences have since been used to further been most useful. However, serological tests sometimes gave define ureaplasma phylogeny (Knox et al., 1998; Kong et al., confusing cross-reactivity patterns and were not ideal for differ- 1999b), as well as to characterize isolates (Knox et al., 1998; entiating strains within a single species (Stemke and Robertson, Knox and Timms, 1998; Kong et al., 1999a; Pitcher et al., 2001). 1985). In an attempt to circumvent problems of cross-reactions The sequences of the 5¢ ends of the mba genes of all 14 serovars obtained with polyclonal antisera, monoclonal antibodies have been defined (Kong et al., 1999a, 2000). Kong et al. (2000) (mAbs) to all 14 serovars of human isolates were developed found that for Ureaplasma parvum, a specific site on the gene (e.g., Echahidi et al., 2001). The use of mAbs coincided with determines serovar identity, whereas for Ureaplasma urealyticum and has been largely overshadowed by the genomic revolution. at least the sequence of the 3¢ end is required and, for certain In the 1980s, the same pattern of partitioning of the ­serovars other serovars, the entire sequence is involved. of human isolates was demonstrated by other phenotypic traits, The genes for the three urease subunits, ureA, ureB, and ureC, traits primarily related to protein structures and functions and adjoining regions from many isolates have been sequenced (Table 138). When phenotypy failed to deliver a clear and (Blanchard, 1990; Kong et al., 1999b; Neyrolles et al., 1996; convenient means of discrimination, genotypy did. The initial Ruifu et al., 1997). Rocha and Blanchard (2002) applied bioin- DNA–DNA relatedness studies of ureaplasmas from humans formatics to predict how certain gene products (e.g., the MBA, (Christiansen et al., 1981) confirmed the two, distinct clusters; restriction and modification systems, transcription anti-termi- however, the large cell biomass, special equipment, and (then) nation elements, and GTP-binding proteins) might exhibit rare expertise required resulted in few strains being tested. New species specificity. PCRs based upon the 16S rRNA genes are techniques, especially PCR, became more easily performed and the most highly conserved, followed by the 16S–23S intergenic less expensive so that more strains were examined and non- region, the urease-associated genes, the mba genes, and the ambiguous results were obtained (Table 139). Supported by region upstream of them. these strong data, the taxonomy of Ureaplasma urealyticum was emended and extended (Robertson et al., 2002). The ten anti- Acknowledgements genic specificities of the larger cluster (known as group 2 or as We thank J. Glass and collaborators at the J. Craig Venter Insti- the T960 biovar) retained the Ureaplasma urealyticum designa- tute and the University of Alabama - Birmingham for the gift tion with strain T960T as the type strain. The remaining four of the genome sequences of Ureaplasma parvum and Ureaplasma antigenic specificities (known as group 1 or the parvo biovar) urealyticum, and J.G. Tully, K.E. Johansson, and C. Williams for were renamed Ureaplasma parvum in recognition of that clus- their suggestions regarding the initial chapter. ter’s considerably smaller genome size; strain 27T, the serovar 3 standard, was designated the type strain. Many PCR primers Differentiation of the species of the genus Ureaplasma to identify species and strains of ureaplasmas have been pub- The first paper on ureaplasma taxonomy stated that “An advan- lished; commercial PCR-based kits are currently available for all tage of forming a new genus is that it confers freedom to classify named Ureaplasma species. new species within the genus without adhering to the principles In summary, current ureaplasma taxonomy is based upon formulated for the other genera. A numbered serovar of a Ure- pragmatic, polyphasic criteria, i.e., a synthesis of phylogeny, aplasma from humans is broadly equivalent to a named species phenotypy, and genotypy. For the specific requirements for within the genera Mycoplasma or Acholeplasma” (Shepard et al., taxonomic studies of Mollicutes, consult the most recent mini- 1974). The serological diversity within the type strain, Ureaplasma mal standards document (Brown et al., 2007). Some ­serological urealyticum, was regarded as no more than antigenic heterogene- testing is mandatory. Rabbit antisera to the 14 serovars of ure- ity, possibly reflecting minor differences within a single epitope. aplasmas of humans and certain animal species are currently To ensure that taxonomy would develop rationally, official rec- available from Jerry K. Davis, Curator of the Mollicutes Collec- ognition of Ureaplasma subspecies was avoided. Thirty-five years tion, School of Veterinary Medicine, Purdue University, West later, this taxonomic restraint can be appreciated. Lafayette, IN, USA. Expertise in determining phenotypic traits When the first genus and species, Ureaplasma urealyticum, specific to ureaplasmas may be accessible through collaboration was named, it had eight known antigenic specificities (Shepard with the appropriate working team of the International Research et al., 1974); 6 years later, the number had reached 14 (Robert- Programme for Comparative Mycoplasmology (IRPCM) of the son and Stemke, 1982) where, surprisingly, it has remained. It International Organization for ­Mycoplasmology (IOM) at www. is surprising because the serotypes were isolated over a 20-year the-iom.org. Genus II. Ureaplasma 621

List of species of the genus Ureaplasma 1. Ureaplasma urealyticum Shepard, Lunceford, Ford, Purcell, ureaplasmas isolated from dogs (represented by the strains Taylor-Robinson, Razin and Black 1974, 167AL emend. Rob- DIM-C, D29M, and D11N-A). The species designation refers ertson, Stemke, Davis, Harasawa, Thirkell, Kong, Shepard only to serogroup I strains, although strain D11N-A shows a and Ford 2002, 593 one-way, serological cross-reaction with D6P-CT. It produces u.re.a.ly¢ti.cum. N.L. fem. n. urea urea; N.L. adj. lyticus -a -um an IgA protease which specifically cleaves canine myeloma (from Gr. adj. lutikos -ê -on) able to loosen, able to dissolve; IgA, but not human or murine IgA. Genome size is 860 kbp T N.L. neut. adj. urealyticum urea-­dissolving or urea-digesting. (PFGE). DNA reassociation values: between D6P-C and the other three canine strains (DIM-C, D29M, and D11N-A) Cells are coccoid and approximately 500 nm in diameter. are 41–63% versus 33% with Ureaplasma urealyticum (strain Coccobacillary forms are seen in exponential phase cultures. T960T). One strain has been shown to have a carbohydrate-­containing Source: habitat is the prepuce, vagina, and oral and nasal capsule; it and others contain lipoglycans. Grows at tempera- cavities of canines. tures between 20 and 40°C, grows better at 30–35°C and best DNA G+C content (mol%): 29.4 (HPLC). at 36–37°C. Colonies are £20–50 mm in diameter with com- Type strain: D6P-C, ATCC 51252, CIP 106087. plete or partial fried-egg morphology. Sequence accession no. (16S rRNA gene): D78648 (type strain). Serologically distinct from all other named species in the genus, but serologically heterogeneous. Ten specific anti- 3. Ureaplasma cati Harasawa, Imada, Ito, Koshimizu, Cassell genic determinants are known: 2, 4, 5, and 7–13. Multiple- and Barile 1990a, 50VP banded antigens are serovar-related and recognized by the ca¢ti. L. gen. n. cati of a cat. host. Like Ureaplasma parvum, it has human IgA-specific pro- tease activity that specifically cleaves human IgA1, but not Cells are coccoid and ³675 nm diameter, exceeding the human IgA2. Has distinctive PAGE and RFLP patterns. DNA 450–550 nm range of most named Ureaplasma species. Coc- is not restricted by endonuclease Uur9601. Genome size of cobacillary forms are seen and occasionally filaments. Col- the type strain is 890 kbp, whereas the sizes of the 10 known onies are £15–140 mm in diameter with diffuse, granular serovar standard strains range from 840 to 1140 kbp (PFGE). appearance; some fried-egg colonies may appear after pas- DNA reassociation values: within the species (serovars 2, 4, saging. Distinct from other established species in the genus, 5, and 7), 69–100%; with Ureaplasma parvum (serovars 1, 3, including Ureaplasma felinum, antigenically and in PAGE and 6), 49–52%. Opportunistic pathogen of humans; causes (Harasawa et al., 1990a) and RFLP patterns (Harasawa et al., some cases of nongonococcal urethritis, infectious kidney 1984). Genome size has not been determined. DNA reasso- stones, systemic infection in immunologically compromised ciation values: 83–100% within feline serogroup SII strains hosts. Associated with a broad variety of urogenital infections (Ureaplasma cati) versus <10% with serogroup SI strains (Ure- for which causality remains to be established. aplasma felinum). Source: primarily found in the genitourinary tract of Source: found in the oral cavity of healthy domestic cats female and male humans; occasionally in the oral cavity and (Felis domestica). rectum. DNA G+C content (mol%): 27.9 (Bd), 28.1 (HPLC) for strain F2T. DNA G+C content (mol%): 25.5–27.8 (Tm; type strain) and 27.7–28.5 (Bd; serovars 2, 4, 5, and 7). Type strain: F2, ATCC 49228, NCTC 11710, CIP 106088. Type strain: T960, (CX8), ATCC 27618, NCTC 10177. Sequence accession nos: D78649 (type strain 16S rRNA gene), Sequence accession nos: M23935 and AF073450 (type strain 16S D63685 (type strain 16S–23S rRNA intergenic spacer region). rRNA gene), AB028088 and AF059330 (type strain 16S–23S 4. Ureaplasma diversum Howard and Gourlay 1982, 450VP rRNA intergenic region). Complete and near-complete (>99%) genomes: serovar 2 strain ATCC 27814, NZ_ABFL00000000; di.ver¢sum. L. neut. part. adj. diversum different, distinct, het- serovar 4 strain ATCC 27816, NZ_AAYO00000000; serovar erogeneous, referring to the difference in polypeptides and 5 strain ATCC 27817, NZ_AAZR00000000; serovar 7 strain G+C content as compared to Ureaplasma urealyticum and to ATCC 27819, NZ_AAYP00000000; serovar 8 strain ATCC the heterogeneous antigenic structure of the species. 27618, NZ_AAYN00000000; serovar 9 strain ATCC 33175, NZ_ Cells are coccoid or coccobacillary and appear to be AAYQ00000000; serovar 10 strain ATCC 33699, NC_011374; within the size range of other named Ureaplasma species serovar 11 strain ATCC 33695, NZ_AAZS00000000; serovar although no measurements have been published. Colonies 12 strain ATCC 33696, NZ_AAZT00000000; serovar 13 strain are £100–175 mm in diameter based on photomicrographs. ATCC 33698, NZ_ABEV00000000. Serologically distinct from other named species but anti- genically heterogeneous, comprising serogroups A, B, and C, 2. Ureaplasma canigenitalium Harasawa, Imada, Kotani, Koshi- and represented by strains A417T, D48, and T44. These show mizu and Barile 1993, 644VP three distinctive PAGE patterns (Howard and Gourlay, 1982), ca.ni.ge.ni.ta¢li.um. L. n. canis dog; L. pl. n. genitalia the geni- but only one RFLP pattern (Harasawa et al., 1984), and, based tals; N.L. pl. gen. n. canigenitalium of canine genitals. upon the latter criterion, were considered homogeneous. Cells are coccoid and about 500 nm in diameter; cocco- Genome size range is 1100–1160 kbp for strains 95 TX, bacillary forms are seen. Colonies are £20–140 mm diameter 1763, and 2065-B202 (PFGE). No DNA reassociation values with fried-egg morphology. Serogroup I strains represented are available. by D6P-CT are serologically distinct from all other established Source: the type strain originated from a pneumonic calf species in the genus and from the other three serogroups of lung. 622 Family I. Mycoplasmataceae

DNA G+C content (mol%): 29.0 and 28.7–30.2 (Bd) for the Source: found only in oropharynx of healthy red jungle type strain and 10 bovine isolates, respectively; thus, higher fowl (Gallus gallus) and chickens (Gallus gallus var. domesticus) than and not overlapping the values for Ureaplasma urealyti- kept as laboratory or zoo animals in Japan and in chickens cum and Ureaplasma parvum. and turkeys with pneumonia or airsacculitis in Hungary. Type strain: A417, ATCC 43321, NCTC 10182, CIP 106089. DNA G+C content (mol%): 27.6 (HPLC). Sequence accession nos: D78650 (type strain 16S rRNA Type strain: D6-1, ATCC 43346, NCTC 11707. gene), D63686 (type strain 16S–23S rRNA intergenic spacer Sequence accession nos: U62937 (type strain 16S rRNA region). gene), D63688 (type strain 16S–23S rRNA intergenic spacer region). 5. Ureaplasma felinum Harasawa, Imada, Ito, Koshimizu, ­Cassell and Barile 1990a, 50VP 7. Ureaplasma parvum Robertson, Stemke, Davis, Harasawa, VP fe.li¢num. L. neut. adj. felinum of or belonging to a cat. Thirkell, Kong, Shepard and Ford 2002, 593 Coccoid cells of ³800 nm diameter exceed the 450–500 nm par¢vum. L. neut. adj. parvum small, referring to its significantly range of most Ureaplasma species. Coccobacillary forms and smaller genome sizes compared to Ureaplasma urealyticum, the occasional filaments are seen. Colonies are £15–140 mm diam- other species from humans. eter with diffuse, granular appearance; some fried-egg colo- Cells are coccoid and about 500 nm in diameter. Cocco- nies may appear after passaging. Distinct antigenically and by bacillary forms are present in exponential phase cultures. PAGE and RFLP patterns (Harasawa et al., 1984) from other Lipoglycans have been identified in a serovar 3 strain. Colo- established species in the genus, including Ureaplasma cati. The nies are £20–140 mm in diameter with complete or partial genome size is 1170 kbp (PFGE). It is the largest of any ure- fried-egg morphology. aplasma strain examined. DNA reassociation values: 89–100% Serologically distinct from all other named species in the within feline serogroup SI strains (Ureaplasma felinum) versus genus, but serological heterogeneity is exhibited within the <10% with serogroup II strains (Ureaplasma cati). species. Four specific antigenic determinants are known: 1, Source: found in the oral cavity of healthy domestic cats 3, 6, and 14. Serovar specificities are related to the multiple- (Felis domestica). banded antigens recognized by the host. Like Ureaplasma DNA G+C content (mol%): 27.9 (HPLC). urealyticum, Ureaplasma parvum has an IgA protease activity Type strain: FT2-B, ATCC 49229, NCTC 11709, CIP that specifically cleaves human IgA1, but not human IgA2. 106090. Distinctive PAGE and RFLP patterns. DNA restricted by Sequence accession nos: D78651 (type strain 16S rRNA gene), endonuclease Uur9601. Genome size: 751,719 kbp for the D63687 (type strain 16S–23S rRNA intergenic spacer region). type strain. Complete genomic sequence of the organism has been reported (Glass et al., 2000). DNA reassociation values: 6. Ureaplasma gallorale Koshimizu, Harasawa, Pan, Kotani, within the species (serovars 1, 3, and 6), 91–102%; with Urea­ Ogata, Stephens and Barile 1987, 337VP plasma urealyticum (serovars 2, 4, 5, 7–13), 38–60%. gal.lo.ra¢le. L. n. gallus a barnyard fowl; L. n. os, oris the Source: primary habitat is the genitourinary tract of female mouth; L. neut. suff. -ale suffix used with the sense of per- and male humans; occasionally found in the oral cavity and taining to; N.L. neut. adj. gallorale relating to the mouth of rectum. As yet, unclear whether an opportunistic pathogen barnyard fowl. in non-gonococcal urethritis, but likely to be so in systemic Cells are coccoid and about 500 nm in diameter; coccoba- infection in immunologically compromised hosts. Associ- cillary forms are seen. Colonies are £15–60 mm in diameter ated with a broad variety of urogenital infections for which with fried-egg morphology. Serologically distinct from all causality remains to be established. other established species in the genus. Isolates have similar DNA G+C content (mol%): 25.5 (from genome sequence)

SDS-PAGE, immunoblot, and RFLP patterns, but demon- and 27.8–28.2 (Tm) for serovars 1 and 6. strate some species heterogeneity based on reassociation val- Type strain: 27, ATCC 27815, NCTC 11736. ues (cluster A strains D6-1T and T9-1; cluster B strain Y8-1). Sequence accession nos: L08642 and AF073456 (type strain Genome size is 760 kbp (PFGE). DNA reassociation values fall 16S rRNA gene), AB028083 and AF059323 (type strain into two clusters: within cluster A (strains D6-1T, D23, F2, F5, 16S–23S rRNA intergenic spacer region). Complete and and T9-1), values are 70–100%; within cluster B (strains Y8-1 near-complete (>99%) genomes: serovar 1 strain ATCC and Y4-2), values are 96–100%; between these clusters, values 27813, NZ_ABES00000000; serovar 3 strain ATCC 27815, are 51–69%. Although these values are below expectations for NC_010503; serovar 3 strain ATCC 700970, NC_002162; a single species status, they exceed the 19–27% reassociation serovar 6 strain ATCC 27818, NZ_AAZQ00000000; serovar values with Ureaplasma urealyticum and Ureaplasma diversum. 14 strain ATCC 33697, NZ_ABER00000000.

References Ajufo, J. and K. Whithear. 1980. The surface layer of Mycoplasma synoviae as demonstrated by the negative staining technique. Res. Vet. Sci. 29: Adler, H.E., J. Fabricant, R. Yamamoto and J. Berg. 1958. Symposium on 268–270. chronic respiratory diseases of poultry. I. Isolation and identification Al-Ankari, A.R. and J.M. Bradbury. 1996. Mycoplasma iowae : a review. of pleuropneumonia-like organisms of avian origin. Am. J. Vet. Res. Avian Pathol. 25: 205–229. 19: 440–447. Al Masalma, M., F. Armougom, W. Scheld, H. Dufour, P. Roche, Adler, S. and V. Ellenbogen. 1934. A note on two new blood parasites of M. Drancourt and D. Raoult. 2009. The expansion of the cattle: Eperythrozoon and Bartonella. J. Comp. Pathol. 47: 220–221. Genus II. Ureaplasma 623

­microbiological spectrum of brain abscesses with use of multiple 16S Bébéar, C. and J.A. Robertson. 1996. Determination of minimal inhibi- ribosomal DNA sequencing. Clin. Infect. Dis. 48: 1169–1178. tory concentration. In Molecular and Diagnostic Procedures in Alberti, A., P. Robino, B. Chessa, S. Rosati, M. Addis, P. Mercier, A. Man- Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, nelli, T. Cubeddu, M. Profiti, E. Bandino, R. Thiery and M. Pittau. New York, pp. 189–197. 2008. Characterisation of Mycoplasma capricolum P60 surface lipopro- Bébéar, C.M. and I. Kempf. 2005. Antimicrobial therapy and antimicro- tein and its evaluation in a recombinant ELISA. Vet. Microbiol. 128: bial resistance. In Mycoplasmas Molecular Biology Pathogenicity and 81–89. Strategies for Control (edited by Blanchard and Browning). Horizon Alexander, P., K. Slee, S. McOrist, L. Ireland and P. Coloe. 1985. Mastitis Bioscience, Norfolk, UK, pp. 535–568. in cows and polyarthritis and pneumonia in calves caused by Myco- Beeton, M., V. Chalker, N. Maxwell, S. Kotecha and O. Spiller. 2009. plasma species bovine group 7. Aust. Vet. J. 62: 135–136. Concurrent titration and determination of antibiotic resistance in Allam, N.M. and R.M. Lemcke. 1975. Mycoplasmas isolated from the Ureaplasma species with identification of novel point mutations in respiratory tract of horses. J. Hyg. (Lond.) 74: 385–407. genes associated with resistance. Antimicrob. Agents Chemother. 53: Amin, M.M. and F.T. Jordan. 1978. Experimental infection of ducklings 2020–2027. with Mycoplasma gallisepticum and Mycoplasma anatis. Res. Vet. Sci. 25: Ben Abdelmoumen Mardassi, B., A.O. Béjaoui Khiari, L., A. Landoulsi, 86–88. C. Brik, B. Mlik and F. Amouna. 2007. Molecular cloning of a Antunes, N., M. Tavío, P. Mercier, R. Ayling, W. Al-Momani, P. Assun- Mycoplasma­ meleagridis-specific antigenic domain endowed with a ção, R. Rosales and J. Poveda. 2007. In vitro susceptibilities of Myco- serodiagnostic potential. Vet. Microbiol. 119: 31–41. plasma putrefaciens field isolates. Antimicrob. Agents Chemother. 51: Bencˇina, D., D. Dorrer and T. Tadina. 1987. Mycoplasma species isolated 3452–3454. from six avian species. Avian Pathol. 16: 653–664. Arif, A., J. Schulz, F. Thiaucourt, A. Taha and S. Hammer. 2007. Conta- Bencˇina, D., T. Tadina and D. Dorrer. 1988. Natural infection of ducks gious caprine pleuropneumonia outbreak in captive wild ungulates with Mycoplasma synoviae and Mycoplasma gallisepticum and Mycoplasma at Al Wabra Wildlife Preservation, State of Qatar. J. Zoo Wildl. Med. egg transmission. Avian Pathol. 17: 441–449. 38: 93–96. Bencˇina, D., I. Mrzel, O. Zorman Rojs, A. Bidovec and A. Dovc. 2003. Armstrong, C.H., M.J. Freeman and L. Sands-Freeman. 1987. Cross- Characterisation of Mycoplasma gallisepticum strains involved in reactions between Mycoplasma hyopneumoniae and Mycoplasma floc- respiratory disease in pheasants and peafowl. Vet. Rec. 152: 230– culare – practical implications for the serodiagnosis of mycoplasmal 234. pneumonia of swine. Isr. J. Med. Sci. 23: 654–656. Berent, L.M. and J.B. Messick. 2003. Physical map and genome sequenc- Armstrong, D., B.H. Yu, A. Yagoda and M.F. Kagnoff. 1971. Coloniza- ing survey of Mycoplasma haemofelis (Haemobartonella felis). Infect. tion of humans by Mycoplasma canis. J. Infect. Dis. 124: 607–609. Immun. 71: 3657–3662. Askaa, G. and H. Erno. 1976. Elevation of Mycoplasma agalactiae subsp. Bergemann, A.D. and L.R. Finch. 1988. Isolation and restriction bovis to species rank, Mycoplasma bovis (Hale et al.) comb. nov. Int. J. endonuclease analysis of a Mycoplasma plasmid. Plasmid 19: Syst. Bacteriol. 26: 323–325. 68–70. Atkinson, T.P., M.F. Balish and K.B. Waites. 2008. Epidemiology, clinical Bergonier, D., X. Berthelot and F. Poumarat. 1997. Contagious agalac- manifestations, pathogenesis and laboratory detection of Mycoplasma tia of small ruminants: current knowledge concerning epidemiology, pneumoniae infections. FEMS Microbiol. Rev. 32: 956–973. diagnosis and control. Rev. Sci. Tech. 16: 848–873. Baker, A.S., K.L. Ruoff and S. Madoff. 1998. Isolation of Mycoplasma Bhugra, B. and K. Dybvig. 1993. Identification and characterization species from a patient with seal finger. Clin. Infect. Dis. 27: 1168– of IS1138, a transposable element from Mycoplasma pulmonis that 1170. belongs to the IS3 family. Mol. Microbiol. 7: 577–584. Balish, M.F. 2006. Subcellular structures of mycoplasmas. Front Biosci. Biberfeld, G. and P. Biberfeld. 1970. Ultrastructural features of Myco- 11: 2017–2027. plasma pneumoniae. J. Bacteriol. 102: 855–861. Balish, M.F. and D.C. Krause. 2006. Mycoplasmas: a distinct cytoskele- Binder, A., R. Aumuller, B. Likitdecharote and H. Kirchhoff. 1990. Isola- ton for wall-less bacteria. J. Mol. Microbiol. Biotechnol. 11: 244–255. tion of Mycoplasma arthritidis from the joint fluid of boars. Zentralbl. Bar-Moshe, B., E. Rapoport and J. Brenner. 1984. Vaccination trials Veterinarmed. B 37: 611–614. against Mycoplasma mycoides subsp. mycoides (large-colony-type) infec- Black, F.T. 1973. Biological and physical properties of human tion in goats. Isr. J. Med. Sci. 20: 972–974. T-mycoplasmas. Ann. N. Y. Acad. Sci. 225: 131–143. Barile, M. 1986. DNA homologies and serologic relationships among Black, F.T. and A. Krogsgaard-Jensen. 1974. Application of indirect ureaplasmas from various hosts. Pediatr. Infect. Dis. 5: S296–299. immunofluorescence, indirect haemagglutination and polyacrylam- Barile, M.F., R.A. Del Giudice, T.R. Carski, C.J. Gibbs and J.A. Morris. ide-gel electrophoresis to human T-mycoplasmas. Acta Pathol. Micro- 1968. Isolation and characterization of Mycoplasma arginini: spec. biol. Scand. [B] Microbiol. Immunol. 82: 345–353. nov. Proc. Soc. Exp. Biol. Med. 129: 489–494. Blanchard, A. 1990. Ureaplasma urealyticum urease genes: use of a UGA Barile, M.F., R.A. Del Giudice and J.G. Tully. 1972. Isolation and charac- tryptophan codon. Mol. Microbiol. 4: 669–676. terization of Mycoplasma conjunctivae sp. n. from sheep and goats with Blanchard, A., W. Hamrick, L. Duffy, K. Baldus and G.H. Cassell. 1993. keratoconjunctivitis. Infect. Immun. 5: 70–76. Use of the polymerase chain reaction for detection of Mycoplasma fer- Barile, M.F. 1984. Immunization against Mycoplasma pneumoniae disease: mentans and Mycoplasma genitalium in the urogenital tract and amni- a review. Isr. J. Med. Sci. 20: 912–915. otic fluid. Clin. Infect. Dis. 17 Suppl 1: S272–279. Baseman, J., M. Cagle, J. Korte, C. Herrera, W. Rasmussen, J. Base- Blanchard, A. 1997. Mycoplasmas and HIV infection, a possible inter- man, R. Shain and J. Piper. 2004. Diagnostic assessment of Myco- action through immune activation. Wien Klin. Wochenschr. 109: plasma genitalium in culture-positive women. J. Clin. Microbiol. 42: 590–593. 203–211. Blanchard, A., L. Montagnier and M.L. Gougeon. 1997. Influence Baseman, J.B., S.F. Dallo, J.G. Tully and D.L. Rose. 1988. Isolation and of microbial infections on the progression of HIV disease. Trends characterization of Mycoplasma genitalium strains from the human Microbiol. 5: 326–331. respiratory tract. J. Clin. Microbiol. 26: 2266–2269. Blanchard, A. and C.M. Bébéar. 2002. Mycoplasmas of Humans. In Baseman, J.B. and J.G. Tully. 1997. Mycoplasmas: sophisticated, re- Molecular Biology and Pathogenicity of Mycoplasmas (edited by emerging, and burdened by their notoriety. Emerg. Infect. Dis. 3: Razin and Herrmann). Kluwer Academic/Plenum Publishers, New 21–32. York, pp. 45–72. 624 Family I. Mycoplasmataceae

Blaylock, M., O. Musatovova, J. Baseman and J. Baseman. 2004. Deter- Brown, D.R., R. Whitcomb and J. Bradbury. 2007. Revised minimal stan- mination of infectious load of Mycoplasma genitalium in clinical sam- dards for description of new species of the class Mollicutes (division ples of human vaginal cells. J. Clin. Microbiol. 42: 746–752. Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Boatman, E.S. 1979. Morphology and ultrastructure of the Mycoplas- Brown, D.R., Clippinger T.L., Helmick K.E., Schumacher I.M., Ben- matales. In The Mycoplasmas, vol. 1 (edited by Barile and Razin). nett R.A., Johnson C.M., Vliet K.A., Jacobson E.R. and B. M.B. 1996. ­Academic Press, New York, pp. 63–102. Mycoplasma isolation during a fatal epizootic of captive alligators Bonilla, H.F., C.E. Chenoweth, J.G. Tully, L.K. Blythe, J.A. Robertson, (Alligator mississippiensis) in Florida. Int. Org. Mycoplasmol. Lett V.M. Ognenovski and C.A. Kauffman. 1997. Mycoplasma felis septic 4: 2. arthritis in a patient with hypogammaglobulinemia. Clin. Infect. Dis. Brown, D.R., J.M. Farley, L.A. Zacher, J.M.R. Carlton, T.L. Clippinger, 24: 222–225. J.G. Tully and M.B. Brown. 2001a. Mycoplasma alligatoris sp. nov., from Boothby, J.T., D.E. Jasper, M.H. Rollins and C.B. Thomas. 1981. Detec- American alligators. Int. J. Syst. Evol. Microbiol. 51: 419–424. tion of Mycoplasma bovis specific IgG in bovine serum by enzyme- Brown, D.R., D.F. Talkington, W.L. Thacker, M.B. Brown, D.L. ­Dillehay linked immunosorbent assay. Am. J. Vet. Res. 42: 1242–1247. and J.G. Tully. 2001b. Mycoplasma microti sp. nov., isolated from the Borrel, A., E. Dujardin-Beaumetz, Jeantet and C. Jouan. 1910. respiratory tract of prairie voles (Microtus ochrogaster). Int. J. Syst. Le microbe de la péripneumonie. Ann. Inst. Pasteur (Paris) 24: Evol. Microbiol. 51: 409–412. 168–179. Brown, D.R., J.L. Merritt, E.R. Jacobson, P.A. Klein, J.G. Tully and M.B. Borup-Christensen, P., K. Erb and J.C. Jensenius. 1988. Curing human Brown. 2004. Mycoplasma testudineum sp. nov., from a desert tortoise hybridomas infected with Mycoplasma hyorhinis. J. Immunol. Methods (Gopherus agassizii) with upper respiratory tract disease. Int. J. Syst. 110: 237–240. Evol. Microbiol. 54: 1527–1529. Boughton, E., S.A. Hopper and P.J.R. Gayford. 1983. Mycoplasma Brown, D.R., L.A. Zacher, L.D. Wendland and M.B. Brown. 2005. Emerg- canadense from bovine fetuses. Vet. Rec. 112: 87. ing Mycoplasmoses in Wildlife. In Mycoplasmas Molecular Biology Bowie, W., S. Wang, E. Alexander, J. Floyd, P. Forsyth, H. Pollock, J. Lin, Pathogenicity and Strategies for Control (edited by Blanchard and T. Buchanan and K. Holmes. 1977. Etiology of nongonococcal ure- Browning). Horizon Bioscience, Norfolk, England, pp. 383–414. thritis. Evidence for Chlamydia trachomatis and Ureaplasma urealyticum. Brown, D.R., D.L. Demcovitz, D.R. Plourde, S.M. Potter, M.E. Hunt, J. Clin. Invest. 59: 735–742. R.D. Jones and D.S. Rotstein. 2006. Mycoplasma iguanae sp. nov., from Bradbury, J.M., F. M. and A. Williams. 1983. Mycoplasma lipofaciens, a a green iguana (Iguana iguana) with vertebral disease. Int. J. Syst. new species of avian origin. International Journal of Systematic and Evol. Microbiol. 56: 761–764. Evolutionary Microbiology 33: 329–335. Brown, D.R. and J.M. Bradbury. 2008. International Committee on Sys- Bradbury, J.M. and M. Forrest. 1984. Mycoplasma cloacale, a new species tematics of Prokaryotes of the International Union of Microbiologi- isolated from a turkey. Int. J. Syst. Bacteriol. 34: 389–392. cal Societies, Subcommittee on the taxonomy of mollicutes, minutes Bradbury, J.M., A. Vuillaume, J.P. Dupiellet, M. Forrest, J.L. Bind and of the meeting, 6 and 11 July 2008, Tianjin, PR China. International G. Gaillardperrin. 1987. Isolation of Mycoplasma cloacale from a Journal of Systematic and Evolutionary Microbiology 58: 2987–2990. number of different avian hosts in Great-Britain and France. Avian Brown, M.B., I.M. Schumacher, P.A. Klein, K. Harris, T. Correll and E.R. Pathol. 16: 183–186. Jacobson. 1994. Mycoplasma agassizii causes upper respiratory tract Bradbury, J.M., F.T.W. Jordan, T. Shimizu, L. Stipkovits and Z. Varga. disease in the desert tortoise. Infect. Immun. 62: 4580–4586. 1988. Mycoplasma anseris sp. nov. found in geese. Int. J. Syst. Bacteriol. Brown, M.B., D.R. Brown, P.A. Klein, G.S. McLaughlin, I.M. ­Schumacher, 38: 74–76. E.R. Jacobson, H.P. Adams and J.G. Tully. 2001c. Mycoplasma agassizii Bradbury, J.M., O.M.S. Abdulwahab, C.A. Yavari, J.P. Dupiellet and J.M. sp. nov., isolated from the upper respiratory tract of the desert tor- Bove. 1993. Mycoplasma imitans sp. nov. is related to Mycoplasma gal- toise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphe- lisepticum and found in birds. Int. J. Syst. Bacteriol. 43: 721–728. mus). Int. J. Syst. Evol. Microbiol. 51: 413–418. Bradbury, J.M., C.M. Dare and C.A. Yavari. 2000. Evidence of Mycoplasma Browning, G.F., K.G. Whithear and S.J. Geary. 2005. Vaccines to Con- gallisepticum in British wild birds. Proceedings of the 13th Congress of trol Mycoplasmosis. In Mycoplasmas: Molecular Biology, Pathogenic- the International Organization for Mycoplasmology Fukouka, Japan, ity, and Strategies for Control (edited by Blanchard and Browning). p. 253. Horizon Bioscience, Norfolk, UK, pp. 569–599. Bradbury, J.M., C.A. Yavari and C.M. Dare. 2001. Mycoplasmas and Brun-Hansen, H., H. Gronstol, H. Waldeland and B. Hoff. 1997. respiratory disease in pheasants and partridges. Avian Pathol. 30: Eperythrozoon­ ovis infection in a commercial flock of sheep. Zentralbl. 391–396. Veterinarmed. B 44: 295–299. Bradbury, J.M. and C.J. Morrow. 2008. Mycoplasma infections. In Poul- Brunner, S., P. Frey-Rindova, M. Altwegg and R. Zbinden. 2000. Ret- try Diseases, 6th edn (edited by Pattison, Bradbury and Alexander). roperitoneal abscess and bacteremia due to Mycoplasma hominis in a Elsevier, Edinburgh, pp. 220–234. polytraumatized man. Infection 28: 46–48. Bradbury, J.M. and S.H. Kleven. 2008. Mycoplasma synoviae infection. Busch, U., H. Nitschko, F. Pfaff, B. Henrich, J. Heesemann and In Diseases of Poultry, 12th edn. (edited by Saif, Fadly, Glisson, M. Abele-Horn. 2000. Molecular comparison of Mycoplasma hominis McDougald, Nolan and Swayne). Blackwell Publishing, Ames, pp. strains isolated from colonized women and women with various uro- 856–864. genital infections. Zentralbl. Bakteriol. 289: 879–888. Brandao, E. 1995. Isolation and identification of Mycoplasma mycoides Buss, I., R. Senthilmohan, B. Darlow, N. Mogridge, A. Kettle and subspecies mycoides SC strains in sheep and goats. Vet. Rec. 136: C. Winterbourn. 2003. 3-Chlorotyrosine as a marker of protein 98–99. damage by myeloperoxidase in tracheal aspirates from preterm Bredt, W. 1979. Motility. In The Mycoplasmas, vol. 1 (edited by Barile infants: association with adverse respiratory outcome. Pediatr. Res. and Razin). Academic Press, New York, pp. 141–155. 53: 455–462. Brown, D.R., G. McLaughlin and M. Brown. 1995. Taxonomy of the Carmichael, L.E., T.D. St George, N.D. Sullivan and N. Horsfall. 1972. feline mycoplasmas Mycoplasma felifaucium, Mycoplasma feliminutum, Isolation, propagation, and characterization studies of an ovine Myco- Mycoplasma felis, Mycoplasma gateae, Mycoplasma leocaptivus, Mycoplasma plasma responsible for proliferative interstitial pneumonia. Cornell leopharyngis, and Mycoplasma simbae by 16S rRNA gene sequence com- Vet. 62: 654–679. parisons. Int. J. Syst. Bacteriol. 45: 560–564. Carson, J.L., P.C. Hu and A.M. Collier. 1992. Cell structure and func- tional elements. In Mycoplasmas: Molecular Biology and Pathogen- Genus II. Ureaplasma 625

esis (edited by Maniloff, McElhaney, Finch, and Baseman). American Cottew, G.S. and F.R. Yeats. 1978. Subdivision of Mycoplasma mycoides Society for Microbiology, Washington, DC, pp. 63–72. subsp. mycoides from cattle and goats into two types. Aust. Vet. J. 54: Casin, I., D. Vexiau-Robert, P. De La Salmoniere, A. Eche, B. ­Grandry 293–296. and M. Janier. 2002. High prevalence of Mycoplasma genitalium Cottew, G.S. 1979. Caprine-ovine mycoplasmas. In The Mycoplasmas, in the lower genitourinary tract of women attending a sexually vol. 2 (edited by Tully and Whitcomb). Academic Press, New York, transmitted disease clinic in Paris, France. Sex. Transm. Dis. 29: pp. 103–132. 353–359. Cottew, G.S. 1983. Recovery and identification of caprine and ovine Cassell, G.H. and A. Hill. 1979. Murine and other small animal myco- mycoplasmas. In Methods in Mycoplasmology, vol. 2 (edited by Tully plasmas. In The Mycoplasmas, vol. 1 (edited by Tully and Whitcomb). and Razin). Academic Press, New York, pp. 91–104. Academic Press, New York, pp. 235–273. Cottew, G.S., A. Breard, A.J. DaMassa, H. Erno, R.H. Leach, P.C. Lefe- Caswell, J.L. and M. Archambault. 2007. Mycoplasma bovis pneumonia in vre, A.W. Rodwell and G.R. Smith. 1987. Taxonomy of the Mycoplasma cattle. Anim. Health Res. Rev. 8: 161–186. mycoides cluster. Isr. J. Med. Sci. 23: 632–635. Chalker, V.J. and J. Brownlie. 2004. Taxonomy of the canine Mollicutes Crespo, R. and R. McMillan. 2008. Facial cellulitis induced in chickens by 16S rRNA gene and 16S/23S rRNA intergenic spacer region by Mycoplasma gallisepticum bacterin and its treatment. Avian Dis. 52: sequence comparison. Int. J. Syst. Evol. Microbiol. 54: 537–542. 698–701. Chalker, V.J. 2005. Canine mycoplasmas. Res. Vet. Sci. 79: 1–8. Daddow, K.N. 1980. Culex annulirostris as a vector of Eperythrozoon ovis Chávez Gonzalez, Y.R., C. Ros Bascunana, G. Bolske, J.G. Mattsson, infection in sheep. Vet. Parasitol. 7: 313–317. C. Fernandez Molina and K.-E. Johansson. 1995. In vitro amplifica- DaMassa, A.J., E.R. Nascimento, M.I. Khan, R. Yamamoto and D.L. tion of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma Brooks. 1991. Characteristics of an unusual Mycoplasma isolated from agalactiae by PCR. Vet. Microbiol. 47: 183–190. a case of caprine mastitis and arthritis with possible systemic manifes- Cheng, X., J. Nicolet, F. Poumarat, J. Regalla, F. Thiaucourt and J. Frey. tations. J. Vet. Diagn. Invest. 3: 55–59. 1995. Insertion element IS1296 in Mycoplasma mycoides subsp. mycoides Dammann, O., E. Allred, D. Genest, R. Kundsin and A. Leviton. 2003. small colony identifies a European clonal line distinct from African Antenatal Mycoplasma infection, the fetal inflammatory response and Australian strains. Microbiology 141: 3221–3228. and cerebral white matter damage in very-low-birthweight infants. Chin, R.P., G.Y. Ghazikhanian and I. Kempf. 2008. Mycoplasma melea- ­Paediatr. Perinat. Epidemiol. 17: 49–57. gridis infection. In Diseases of Poultry, 12th edn. (edited by Saif, Damassa, A.J., J.G. Tully, D.L. Rose, D. Pitcher, R.H. Leach and G.S. Fadly, Glisson, McDougald, Nolan and Swayne). Blackwell Publish- Cottew. 1994. Mycoplasma auris sp. nov., Mycoplasma cottewii sp. nov., ing, Ames, pp. 834–845. and Mycoplasma yeatsii sp. nov., new sterol-requiring mollicutes from Christiansen, C., F.T. Black and E.A. Freundt. 1981. Hybridization the external ear canals of goats. Int. J. Syst. Bacteriol. 44: 479–484. experiments with DNA from Ureaplasma urealyticum, serovars I to VIII. DaMassa, A.J. 1996. Mycoplasma infections of goat and sheep. In Molecu- Int. J. Syst. Bacteriol. 31: 259–262. lar and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Clapper, B., A.H. Tu, W.L. Simmons and K. Dybvig. 2004. Bacteriophage Tully and Razin). Academic Press, San Diego, pp. 265–273. MAV1 is not associated with virulence of Mycoplasma arthritidis. Infect. Davis, J.W., Jr., I.S. Moses, C. Ndubuka and R. Ortiz. 1987. Inorganic Immun. 72: 7322–7325. pyrophosphatase activity in cell-free extracts of Ureaplasma urealyti- Clark, H.W., J. S. Bailey, D. C. Laughlin and T.M. Brown. 1978. Isolation cum. J. Gen. Microbiol. 133: 1453–1459. of Mycoplasma from the genital tracts of elephants. Zentralbl. Bakte- Davis, J.W., Jr. and I. Villanueva. 1990. Enzyme differences in serovar riol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 241:262. clusters of Ureaplasma urealyticum. In Recent Advances in Mycoplas- Clark, H.W., D. C. Laughlin, J. S. Bailey and T.M. Brown. 1980. Myco- mology (edited by Stanek, Cassell, Tully and Whitcomb). Gustav plasma Species and Arthritis in Captive Elephants. Journal of Zoo ­Fischer Verlag, New York, pp. 665–666. Animal Medicine 11: 3–15. de Barbeyrac, B., C. Bernet-Poggi, F. Fébrer, H. Renaudin, M. Dupon Clark, R. 1942. Eperythrozoon felis (sp. nov.) in a cat. J. Afr. Vet. Med. and C. Bébéar. 1993. Detection of Mycoplasma pneumoniae and Myco- Assoc. 13: 15–16. plasma genitalium in clinical samples by polymerase chain reaction. Cobb, D.T., D.H. Ley and P.D. Doerr. 1992. Isolation of Mycoplasma Clin. Infect. Dis. 17 Suppl 1: S83–89. gallopavonis from free-ranging wild turkeys in coastal North Caro- De Francesco, M., R. Negrini, G. Pinsi, L. Peroni and N. Manca. 2009. lina seropositive and culture-negative for Mycoplasma gallisepticum. Detection of Ureaplasma biovars and polymerase chain reaction- J. Wildl. Dis. 28: 105–109. based subtyping of Ureaplasma parvum in women with or without Cocks, B., F. Brake, A. Mitchell and L. Finch. 1985. Enzymes of inter- symptoms of genital infections. Eur. J. Clin. Microbiol. Infect. Dis. mediary carbohydrate metabolism in Ureaplasma urealyticum and 28: 641–646. Mycoplasma mycoides subsp. mycoides. J. Gen. Microbiol. 131: 2129– de la Fe, C., P. Assunção, P. Saavedra, S. Tola, C. Poveda and J. Poveda. 2135. 2007. Vaccine. Field trial of two dual vaccines against Mycoplasma aga- Cocks, B.G. and L.R. Finch. 1987. Characterization of a restriction lactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in endonuclease from Ureaplasma urealyticum 960 and differences in goats 25: 2340–2345. deoxyribonucleic acid modification of human ureaplasmas. Int. J. De Silva, N. and P. Quinn. 1986. Endogenous activity of phospholi- Syst. Bacteriol. 37: 451–453. pases A and C in Ureaplasma urealyticum. J. Clin. Microbiol. 23: Cole, B.C., L. Golightly and J.R. Ward. 1967. Characterization of Myco- 354–359. plasma strains from cats. J. Bacteriol. 94: 1451–1458. Deeb, B. and G. Kenny. 1967. Characterization of Mycoplasma pulmonis Cole, B.C., L.R. Washburn and D. Taylor-Robinson. 1985. Mycoplasma- variants isolated from rabbits. I. Identification and properties of iso- induced arthritis. In The Mycoplasmas, vol. 4 (edited by Razin and lates. J. Bacteriol. 93: 1416–1424. Barile). Academic Press, New York, pp. 108–160. Del Giudice, R.A., T.R. Carski, M.F. Barile, R.M. Lemcke and J.G. Tully. Cole, R.M. 1983. Transmission electron microscopy: Basic techniques. 1971. Proposal for classifying human strain Navel and related ­simian In Methods in Mycoplasmology (edited by Razin and Tully). Aca- mycoplasmas as Mycoplasma primatum sp. n. J. Bacteriol. 108: 439–445. demic Press, New York, p. 4350. Del Giudice, R.A., R.H. Purcell, T.R. Carski and R.M. Chanock. 1974. Cordtz, J. and J. Jensen. 2006. Disseminated Ureaplasma urealyticum Mycoplasma lipophilum sp. nov. Int. J. Syst. Bacteriol. 24: 147–153. infection in a hypo-gammaglobulinaemic renal transplant patient. Scand. J. Infect. Dis. 38: 1114–1117. 626 Family I. Mycoplasmataceae

Del Giudice, R.A., J.G. Tully, D.L. Rose and R.M. Cole. 1985. Mycoplasma Edward, D.G. and E.A. Freundt. 1969. Proposal for classifying organ- pirum sp. nov., a terminal structured mollicute from cell cultures. Int. isms related to Mycoplasma laidlawii in a family Sapromycetaceae, genus J. Syst. Bacteriol. 35: 285–291. Sapromyces, within the Mycoplasmatales. J. Gen. Microbiol. 57: 391– Del Giudice, R.A., D.L. Rose and J.G. Tully. 1995. Mycoplasma adleri sp. 395. nov., an isolate from a goat. Int. J. Syst. Bacteriol. 45: 29–31. Edward, D.G. and E.A. Freundt. 1970. Amended nomenclature for Del Giudice, R.A. and R.S. Gardella. 1996. Antibiotic treatment of strains related to Mycoplasma laidlawii. J. Gen. Microbiol. 62: 1–2. mycoplasma-infected cell cultures. In Molecular and Diagnostic Edward, D.G.f. 1953. Organisms of the pleuropneumonia group caus- ­Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). ing disease in goats. Vet. Rec. 63: 873–874. Academic Press, San Diego, pp. 439–443. Edward, D.G.f. 1955. A suggested classification and nomenclature Dessì, D., P. Rappelli, N. Diaz, P. Cappuccinelli and P.L. Fiori. 2006. for organisms of the pleuropneumonia group. Int. Bull. Bacteriol. Mycoplasma hominis and Trichomonas vaginalis: a unique case of sym- Nomencl. Taxon. 5: 85–93. biotic relationship between two obligate human parasites. Front. Egwu, G., J. Ameh, M. Aliyu and F. Mohammed. 2001. Caprine Myco- ­Biosci. 11: 2028–2034. plasmal mastitis in Nigeria. Small Rumin. Res. 39: 87–91. Dhondt, A.A., S. Altizer, E.G. Cooch, A.K. Davis, A. Dobson, M.J. Driscoll, Erdélyi, K., M. Tenk and A. Dan. 1999. Mycoplasmosis associated pero- B.K. Hartup, D.M. Hawley, W.M. Hochachka, P.R. Hosseini, C.S. sis type skeletal deformity in a saker falcon nestling in Hungary. J. Jennelle, G.V. Kollias, D.H. Ley, E.C. Swarthout and K.V. Syden- Wildl. Dis. 35: 586–590. stricker. 2005. Dynamics of a novel pathogen in an avian host: Myco- Erickson, B.Z., R.F. Ross, D.L. Rose, J.G. Tully and J.M. Bové. 1986. plasmal conjunctivitis in house finches. Acta Trop. 94: 77–93. Mycoplasma hyopharyngis, a new species from swine. Int. J. Syst. Bac- Dhondt, A.A., K.V. Dhondt, D.M. Hawley and C.S. Jennelle. 2007. teriol. 36: 55–59. Experimental evidence for transmission of Mycoplasma gallisepticum Erickson, B.Z., R.F. Ross and J.M. Bove. 1988. Isolation of Mycoplasma in house finches by fomites. Avian Pathol. 36: 205–208. salivarium from swine. Vet. Microbiol. 16: 385–390. Dierks, R.E., J.A. Newman and B.S. Pomeroy. 1967. Characterization of Evans-Davis, K.D., D.L. Dillehay, D.N. Wargo, S.K. Webb, D.F. Talk- avian Mycoplasma. Ann. N. Y. Acad. Sci. 143: 170–189. ington, W.L. Thacker, L.S. Small and M.B. Brown. 1998. Patho- Dillehay, D.L., M. Sander, D.F. Talkington, W.L. Thacker and genicity of Mycoplasma volis in mice and rats. Lab. Anim. Sci. 48: D.R. Brown. 1995. Isolation of mycoplasmas from prairie voles 38–44. (Microtus ochrogaster). Lab. Anim. Sci. 45: 631–634. Feberwee, A., J. de Wit and W. Landman. 2009. Induction of eggshell Djordjevic, S.R., W.A. Forbes, J. Forbes-Faulkner, P. Kuhnert, S. Hum, apex abnormalities by Mycoplasma synoviae: field and experimental M.A. Hornitzky, E.M. Vilei and J. Frey. 2001. Genetic diversity among studies. Avian Pathol. 38: 77–85. Mycoplasma species bovine group 7: clonal isolates from an outbreak Ferguson, N.M., V.A. Leiting and S.H. Klevena. 2004. Safety and efficacy of polyarthritis, mastitis, and abortion in dairy cattle. Electrophoresis of the avirulent Mycoplasma gallisepticum strain K5054 as a live vaccine 22: 3551–3561. in poultry. Avian Dis. 48: 91–99. Doig, P.A. 1981. Bovine genital mycoplasmosis. Can. Vet. J. 22: 339–343. Fernandez Guerrero, M.L., J. Manuel Ramos and F. Soriano. 1999. Myco- dos Santos, A.P., R.P. dos Santos, A.W. Biondo, J.M. Dora, L.Z. plasma hominis bacteraemia not associated with genital infections. J. Goldani, S.T. de Oliveira, A.M. de Sa Guimaraes, J. Timenetsky, Infect. 39: 91–94. H.A. de Morais, F.H. Gonzalez and J.B. Messick. 2008. Hemoplasma Ferrell, R., M. Heidari, K. Wise and M. McIntosh. 1989. A Mycoplasma infection in HIV-positive patient, Brazil. Emerg Infect Dis 14: 1922– genetic element resembling prokaryotic insertion sequences. Mol. 1924. Microbiol. 3: 957–967. Dowers, K.L., C. Olver, S.V. Radecki and M.R. Lappin. 2002. Use Fischer, R.S., B.E. Fischer and R.A. Jensen. 1992. Sources of amino of enrofloxacin for treatment of large-form Haemobartonella felis acids. In Mycoplasmas: Molecular Biology and Pathogenesis (edited in experimentally infected cats. J. Am. Vet. Med. Assoc. 221: by Maniloff, McElhaney, Finch and Baseman). American Society for 250–253. Microbiology, Washington, D.C., pp. 201–209. Drexler, H.G. and C.C. Uphoff. 2002. Mycoplasma contamination of cell Fitzmaurice, J., M. Sewell, L. Manso-Silvan, F. Thiaucourt, W.L. cultures: Incidence, sources, effects, detection, elimination, preven- ­McDonald and J.S. O’Keefe. 2008. Real-time polymerase chain reac- tion. Cytotechnology 39: 75–90. tion assays for the detection of members of the Mycoplasma mycoides Dubosson, C.R., C. Conzelmann, R. Miserez, P. Boerlin, J. Frey, W. cluster. N Z Vet. J. 56: 40–47. Zimmermann, H. Hani and P. Kuhnert. 2004. Development of two Flint, J.C. and McKelvie D.H. 1956. Feline infectious anemia-diagnosis real-time PCR assays for the detection of Mycoplasma hyopneumoniae and treatment. Presented at the Proc. 92nd Ann. Meet. Amer. Vet. in clinical samples. Vet. Microbiol. 102: 55–65. Med. Assoc. 1955, 240–242. Duffy, L., J. Glass, G. Hall, R. Avery, R. Rackley, S. Peterson and Foley, J.E. and N.C. Pedersen. 2001. “Candidatus Mycoplasma haemo- K. Waites. 2006. Fluoroquinolone resistance in Ureaplasma parvum in minutum”, a low-virulence epierythrocytic parasite of cats. Int. J. Syst. the United States. J. Clin. Microbiol. 44: 1590–1591. Evol. Microbiol. 51: 815–817. Dupiellet, J.-P. 1984. Mycoplasmes et acholeplasmes des palmipedes a Ford, D. 1967. Relationships between Mycoplasma and the etiology of foie gras: isolement, caracterisation, etude du role dans la patholo- nongonococcal urethritis and Reiter’s syndrome. Ann. N. Y. Acad. gie. Universite de Bordeaux II. Sci. 143: 501–504. Dyer, N., L. Hansen-Lardy, D. Krogh, L. Schaan and E. Schamber. 2008. Ford, D. and J. MacDonald. 1967. Influence of urea on the growth of An outbreak of chronic pneumonia and polyarthritis syndrome T-strain mycoplasmas. J. Bacteriol. 93: 1509–1512. caused by Mycoplasma bovis in feedlot bison (Bison bison). J. Vet. Ford, D. 1972. Inhibition of growth of T-strain mycoplasmas by Diagn. Invest. 20: 369–371. hydroxamic acids and by aurothiomalate. Antimicrob Agents Echahidi, F., G. Muyldermans, S. Lauwers and A. Naessens. 2001. Devel- Chemother 2: 340–343. opment of an enzyme-linked immunosorbent assay for serotyping Ford, D. and J. Smith. 1974. Non-specific urethritis associated with a Ureaplasma urealyticum strains using monoclonal antibodies. Clin. tetracycline-resistant T-mycoplasma. Br. J. Vener. Dis. 50: 373–374. Diagn. Lab. Immunol. 8: 52–57. Forrest, M. and J.M. Bradbury. 1984. Mycoplasma glycophilum, a new spe- Edward, D.G. and A.D. Kanarek. 1960. Organisms of the pleuropneu- cies of avian origin. J. Gen. Microbiol. 130: 597–603. monia group of avian origin: their classification into species. Ann. N. Forrest, M. and J. M. Bradbury. 1984. Mycoplasma glycophilum sp. nov. Y. Acad. Sci. 79: 696–702. Validation List No. 15. Int. J. Syst. Bacteriol. 34: 355–357. Genus II. Ureaplasma 627

Forsyth, M.H., J.G. Tully, T.S. Gorton, L. Hinckley, S. Frasca, Jr., H.J. the prevention of avian respiratory mycoplasmosis. Vaccine 26: van Kruiningen and S.J. Geary. 1996. Mycoplasma sturni sp. nov., from 2010–2019. the conjunctiva of a European starling (Sturnus vulgaris). Int. J. Syst. Gautier-Bouchardon, A.V., A.K. Reinhardt, M. Kobisch and I. Kempf. Bacteriol. 46: 716–719. 2002. In vitro development of resistance to enrofloxacin, erythro- Frasca, S., Jr, L. Hinckley, M. Forsyth, T. Gorton, S. Geary and H. Van mycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisep- Kruiningen. 1997. Mycoplasmal conjunctivitis in a European star- ticum, Mycoplasma iowae and Mycoplasma synoviae. Vet. Microbiol. 88: ling. J. Wildl. Dis. 33: 336–339. 47–58. Frasca, S., Jr, E. Weber, H. Urquhart, X. Liao, M. Gladd, K. Cecchini, P. George, J.W., B.A. Rideout, S.M. Griffey and N.C. Pedersen. 2002. Effect Hudson, M. May, R. Gast, T. Gorton and S. Geary. 2005. Isolation and of preexisting FeLV infection or FeLV and feline immunodeficiency characterization of Mycoplasma sphenisci sp. nov. from the choana of virus coinfection on pathogenicity of the small variant of Haemobarto- an aquarium-reared jackass penguin (Spheniscus demersus). J. Clin. nella felis in cats. Am. J. Vet. Res. 63: 1172–1178. Microbiol. 43: 2976–2979. Geraci, J.R., D.J. Staubin, I.K. Barker, R.G. Webster, V.S. Hinshaw, W.J. Freundt, E.A. 1953. The occurrence of Micromyces ­(pleuropneumonia- Bean, H.L. Ruhnke, J.H. Prescott, G. Early, A.S. Baker, S. Madoff and like organisms) in the female genito-urinary tract. Acta Pathol. R.T. Schooley. 1982. Mass mortality of harbor seals: pneumonia asso- Microbiol. Scand. 32: 468–480. ciated with influenza A virus. Science 215: 1129–1131. Freundt, E.A. 1955. The classification of the pleuropneumoniae group Gerchman, I., I. Lysnyansky, S. Perk and S. Levisohn. 2008. In vitro sus- of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. ceptibilities to fluoroquinolones in current and archived Mycoplasma Taxon. 5: 67–78. gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys. Freundt, E.A., Taylorro.D, R.H. Purcell, R.M. Chanock and F.T. Black. Vet. Microbiol. 131: 266–276. 1974. Proposal of Mycoplasma buccale nom. nov. and Mycoplasma fau- Germain, M., M.A. Krohn, S.L. Hillier and D.A. Eschenbach. 1994. Gen- cium nom. nov. for Mycoplasma orale types 2 and 3, respectively. Int. J. ital flora in pregnancy and its association with intrauterine growth Syst. Bacteriol. 24: 252–255. retardation. J. Clin. Microbiol. 32: 2162–2168. Frey, J., X. Cheng, P. Kuhnert and J. Nicolet. 1995. Identification and Gibson, D.G., G.A. Benders, C. Andrews-Pfannkoch, E.A. Denisova, H. characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC Baden-Tillson, J. Zaveri, T.B. Stockwell, A. Brownley, D.W. Thomas, and presence in related mycoplasmas. Gene 160: 95–100. M.A. Algire, C. Merryman, L. Young, V.N. Noskov, J.I. Glass, J.C. Ven- Frey, M.L., R.P. Hanson and D.P. Anderson. 1968. A medium for the ter, C.A. Hutchison, 3rd and H.O. Smith. 2008. Complete chemical isolation of avian mycoplasmas. American Journal of Veterinary synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Research 29: 2163–2171. Science 319: 1215–1220. Friis, N.F. and E. Blom. 1983. Isolation of Mycoplasma canadense from Giebel, J., A. Binder and H. Kirchhoff. 1990. Isolation of Mycoplasma bull semen. Acta Vet. Scand. 24: 315–317. moatsii from the intestine of wild Norway rats (Rattus norvegicus). Friis, N.F. and J. Szancer. 1994. Sensitivity of certain porcine and bovine Vet. Microbiol. 22: 23–29. mycoplasmas to antimicrobial agents in a liquid medium test com- Giebel, J., J. Meier, A. Binder, J. Flossdorf, J.B. Poveda, R. Schmidt and pared to a disc assay. Acta Vet. Scand. 35: 389–394. H. Kirchhoff. 1991. Mycoplasma phocarhinis sp. nov. and Mycoplasma Furness, G. 1973. T-mycoplasmas: their growth and production of a phocacerebrale sp. nov., two new species from harbor seals (Phoca vitu- toxic substance in broth. J. Infect. Dis. 127: 9–16. lina L). Int. J. Syst. Bacteriol. 41: 39–44. Furness, G. 1975. T-mycoplasmas: Growth patterns and physical charac- Gil, M.C., F.J. Pena, J. Hermoso De Mendoza and L. Gomez. 2003. Gen- teristics of some human strains. J. Infect. Dis. 132: 592–596. ital lesions in an outbreak of caprine contagious agalactia caused Furr, P.M., D. Taylor-Robinson and A.D. Webster. 1994. Mycoplasmas by Mycoplasma agalactiae and Mycoplasma putrefaciens. J. Vet. Med. B and ureaplasmas in patients with hypogammaglobulinaemia and Infect. Dis. Vet. Public Health 50: 484–487. their role in arthritis: microbiological observations over twenty years. Glass, J., E. Lefkowitz, J. Glass, C. Heiner, E. Chen and G. Cassell. 2000. Ann. Rheum. Dis. 53: 183–187. The complete sequence of the mucosal pathogen Ureaplasma urealyti- Gallagher, J. and K. Rhoades. 1983. Scanning electron and light microscopy cum. Nature 407: 757–762. of selected avian strains of Mycoplasma iowae. Avian Dis. 27: 211–217. Glass, J.I., B.A. Methe, V. Paralanov, L.B. Duffy and K.B. Waites. 2008. Gambini, D., I. Decleva, L. Lupica, M. Ghislanzoni, M. Cusini and Comparative genome analysis of all 14 Ureaplasma parvum and E. Alessi. 2000. Mycoplasma genitalium in males with nongonococ- Ureaplasma­ urealyticum serovars. Presented at the International Orga- cal urethritis: prevalence and clinical efficacy of eradication. Sex. nization for Mycoplasmology, Tianjin, China. Transm. Dis. 27: 226–229. Goldberg, D.R., M.D. Samuel, C.B. Thomas, P. Sharp, G.L. Krapu, J.R. Ganapathy, K. and J.M. Bradbury. 1998. Pathogenicity of Mycoplasma Robb, K.P. Kenow, C.E. Korschgen, W.H. Chipley, M.J. Conroy and gallisepticum and Mycoplasma imitans in red-legged partridges (Alecto- et al. 1995. The occurrence of mycoplasmas in selected wild North ris rufa). Avian Pathol. 27: 455–463. American waterfowl. J. Wildl. Dis. 31: 364–371. García-Castillo, M., M. Morosini, M. Gálvez, F. Baquero, R. del Campo Gonçalves, L.F., T. Chaiworapongsa and R. Romero. 2002. Intrauterine and M. Meseguer. 2008. Differences in biofilm development and infection and prematurity. Ment. Retard. Dev. Disabil. Res. Rev. 8: 3–13. antibiotic susceptibility among clinical Ureaplasma urealyticum and Ure- Goodison, S., K. Nakamura, K.A. Iczkowski, S. Anai, S.K. Boehlein and aplasma parvum isolates. J. Antimicrob. Chemother. 62: 1027–1030. C.J. Rosser. 2007. Exogenous mycoplasmal p37 protein alters gene Garcia-Porrua, C., F.J. Blanco, A. Hernandez, A. Atanes, F. Galdo, R. expression, growth and morphology of prostate cancer cells. Cyto- Moure and A. Alonso. 1997. Septic arthritis by Mycoplasma hominis: a genet Genome Res. 118: 204–213. case report and review of the medical literature. Ann. Rheum. Dis. Goodwin, R.F.W., A.P. Pomeroy and P. Whittlestone. 1965. Production 56: 699–700. of enzootic pneumonia in pigs with a Mycoplasma. Vet. Rec. 77: 1247– Gass, R., J. Fisher, D. Badesch, M. Zamora, A. Weinberg, H. Melsness, 1249. F. Grover, J.G. Tully and F.C. Fang. 1996. Donor-to-host transmission Gorton, T.S., M.M. Barnett, T. Gull, R.A. French, Z. Lu, G.F. Kutish, of Mycoplasma hominis in lung allograft recipients. Clin. Infect. Dis. L.G. Adams and S.J. Geary. 2005. Development of real-time diagnos- 22: 567–568. tic assays specific for Mycoplasma mycoides subspecies mycoides Small Gates, A.E., S. Frasca, A. Nyaoke, T.S. Gorton, L.K. Silbart and Colony. Vet. Microbiol. 111: 51–58. S.J. Geary. 2008. Comparative assessment of a metabolically attenuated Mycoplasma gallisepticum mutant as a live vaccine for 628 Family I. Mycoplasmataceae

Gourlay, R.N. and R.H. Leach. 1970. A new Mycoplasma species isolated Harasawa, R., Y. Imada, M. Ito, K. Koshimizu, G.H. Cassell and from pneumonic lungs of calves (Mycoplasma dispar sp. nov.). J. Med. M.F. Barile. 1990a. Ureaplasma felinum sp. nov. and Ureaplasma cati sp. nov. Microbiol. 3: 111–123. isolated from the oral cavities of cats. Int. J. Syst. Bacteriol. 40: 45–51. Gourlay, R.N., R.H. Leach and C.J. Howard. 1974. Mycoplasma verecun- Harasawa, R., E.B. Stephens, K. Koshimizu, I.J. Pan and M.F. Barile. dum, a new species isolated from bovine eyes. J. Gen. Microbiol. 81: 1990b. DNA relatedness among established Ureaplasma species and 475–484. unidentified feline and canine serogroups. Int. J. Syst. Bacteriol. 40: Gourlay, R.N., S.G. Wyld and R.H. Leach. 1977. Mycoplasma alvi, a new 52–55. species from bovine intestinal and urogenital tracts. Int. J. Syst. Bac- Harasawa, R., K. Dybvig, H.L. Watson and G.H. Cassell. 1991. Two teriol. 27: 86–96. genomic clusters among 14 serovars of Ureaplasma urealyticum. Syst. Gourlay, R.N., S.G. Wyld and R.H. Leach. 1978. Mycoplasma sualvi, a Appl. Microbiol. 14: 393–396. new species from intestinal and urogenital tracts of pigs. Int. J. Syst. Harasawa, R., Y. Imada, H. Kotani, K. Koshimizu and M.F. Barile. 1993. Bacteriol. 28: 289–292. Ureaplasma canigenitalium sp. nov., isolated from dogs. Int. J. Syst. Bac- Gourlay, R.N. and C.J. Howard. 1979. Bovine mycoplasmas. In The teriol. 43: 640–644. Mycoplasmas, vol. 2 (edited by Tully and Whitcomb). Academic Harasawa, R. and Y. Kanamoto. 1999. Differentiation of two biovars of Press, New York, pp. 49–102. Ureaplasma urealyticum based on the 16S-23S rRNA intergenic spacer Gourlay, R.N., S.G. Wyld and M.E. Poulton. 1983. Some characteristics region. J. Clin. Microbiol. 37: 4135–4138. of Mycoplasma virus Hr 1, isolated from and infecting Mycoplasma Harasawa, R., D.G. Pitcher, A.S. Ramirez and J.M. Bradbury. 2004. A hyorhinis. Brief report. Arch. Virol. 77: 81–85. putative transposase gene in the 16S-23S rRNA intergenic spacer Grattard, F., B. Pozzetto, B. de Barbeyrac, H. Renaudin, M. Clerc, O. region of Mycoplasma imitans. Microbiology 150: 1023–1029. Gaudin and C. Bébéar. 1995. Arbitrarily-primed PCR confirms the Hasselbring, B.M., J.L. Jordan, R.W. Krause and D.C. Krause. 2006. differentiation of strains of Ureaplasma urealyticum into two biovars. Terminal organelle development in the cell wall-less bacterium Mol. Cell Probes 9: 383–389. Mycoplasma pneumoniae. Proc. Natl. Acad. Sci. USA 103: 16478– Grau, O., F. Laigret, P. Carle, J. Tully, D. Rose and J. Bové. 1991. Identi- 16483. fication of a plant-derived mollicute as a strain of an avian pathogen, Hatchel, J., R. Balish, M. Duley and M. Balish. 2006. Ultrastructure and Mycoplasma iowae, and its implications for mollicute taxonomy. Int. J. gliding motility of Mycoplasma amphoriforme, a possible human respi- Syst. Bacteriol. 41: 473–478. ratory pathogen. Microbiology 152: 2181–2189. Gray, L.D., K.L. Ketring and Y.W. Tang. 2005. Clinical use of 16S Hatchel, J.M. and M.F. Balish. 2008. Attachment organelle ultrastruc- rRNA gene sequencing to identify Mycoplasma felis and M. gateae ture correlates with phylogeny, not gliding motility properties, in associated with feline ulcerative keratitis. J. Clin. Microbiol. 43: Mycoplasma pneumoniae relatives. Microbiology 154: 286–295. 3431–3434. Haulena, M., F. Gulland, J. Lawrence, D. Fauquier, S. Jang, B. Aldridge, Greco, G., M. Corrente, V. Martella, A. Pratelli and D. Buonavoglia. T. Spraker, L. Thomas, D. Brown, L. Wendland and M. Davidson. 2001. A multiplex-PCR for the diagnosis of contagious agalactia of 2006. Lesions associated with a novel Mycoplasma sp. in California sea sheep and goats. Mol. Cell. Probes 15: 21–25. lions (Zalophus californianus) undergoing rehabilitation. J. Wildl. Green III, F. and R.P. Hanson. 1973. Ultrastructure and capsule of Myco- Dis. 42: 40–45. plasma meleagridis. Journal of Bacteriology 116: 1011–1018. Heggie, A.D., D. Bar-Shain, B. Boxerbaum, A.A. Fanaroff, M.A. Grenabo, L., H. Hedelin and S. Pettersson. 1988. Urinary infection O’Riordan and J.A. Robertson. 2001. Identification and quantifica- stones caused by Ureaplasma urealyticum: a review. Scand. J. Infect. tion of ureaplasmas colonizing the respiratory tract and assessment Dis. Suppl. 53: 46–49. of their role in the development of chronic lung disease in preterm Grisold, A., M. Hoenigl, E. Leitner, K. Jakse, G. Feierl, R. Raggam and infants. Pediatr. Infect. Dis. J. 20: 854–859. E. Marth. 2008. Submasseteric abscess caused by Mycoplasma salivar- Henderson, G. and G. Jensen. 2006. Three-dimensional structure of ium infection. J. Clin. Microbiol. 46: 3860–3862. Mycoplasma pneumoniae’s attachment organelle and a model for its Groebel, K., K. Hoelzle, M.M. Wittenbrink, U. Ziegler and L.E. Hoelzle. role in gliding motility. Mol. Microbiol. 60: 376–385. 2009. Mycoplasma suis invades porcine erythrocytes. Infect. Immun. Heyward, J.T., M.Z. Sabry and W.R. Dowdle. 1969. Characterization of 77: 576–584. Mycoplasma species of feline origin. Am. J. Vet. Res. 30: 615–622. Gwaltney, S.M. and R.D. Oberst. 1994. Comparison of an improved Hill, A. 1971. Mycoplasma caviae, a new species. J. Gen. Microbiol. 65: polymerase chain reaction protocol and the indirect hemaggluti- 109–113. nation assay in the detection of Eperythrozoon suis infection. J. Vet. Hill, A. 1977. The isolation of mycoplasmas from non-human primates. Diagn. Invest. 6: 321–325. Vet. Rec. 101: 117. Hale, H.H., C.F. Helmboldt, W.N. Plastridge and E.F. Stula. 1962. Hill, A.C. 1983a. Mycoplasma cricetuli, a new species from the conjuncti- Bovine mastitis caused by a Mycoplasma species. Cornell Vet. 52: vas of chinese hamsters. Int. J. Syst. Bacteriol. 33: 113–117. 582–591. Hill, A.C. 1983b. Mycoplasma collis, a new species isolated from rats and Hammond, P., A. Ramírez, C. Morrow and J. Bradbury. 2009. Devel- mice. Int. J. Syst. Bacteriol. 33: 847–851. opment and evaluation of an improved diagnostic PCR for Myco- Hill, A.C. 1984. Mycoplasma cavipharyngis, a new species isolated from plasma synoviae using primers located in the haemagglutinin the nasopharynx of guinea pigs. J. Gen. Microbiol. 130: 3183–3188. encoding gene vlhA and its value for strain typing. Vet. Microbiol. Hill, A.C. 1985. Mycoplasma testudinis, a new species isolated from a tor- 136: 61–68. toise. Int. J. Syst. Bacteriol. 35: 489–492. Hannan, P. 2000. Guidelines and recommendations for antimicrobial Hill, A.C. 1986. Mycoplasma felifaucium, a new species isolated from the minimum inhibitory concentration (MIC) testing against veterinary respiratory tract of pumas. J. Gen. Microbiol. 132: 1923–1928. Mycoplasma species. International Research Programme on Compar- Hill, A.C. 1988. In Validation of the publication of new names and new ative Mycoplasmology. Vet. Res. 31: 373–395. combinations previously effectively published outside the IJSB. List Harasawa, R., K. Koshimizu, I. Pan, E. Stephens and M. Barile. 1984. no. 27. Int. J. Syst. Bacteriol. 38: 449. Genomic analysis of avian and feline Ureaplasmas by restriction endo- Hill, A.C. 1989. In Validation of the publication of new names and new nucleases. Isr. J. Med. Sci. 20: 942–945. combinations previously effectively published outside the IJSB. List Harasawa, R., K. Koshimizu, I. Pan and M. Barile. 1985. Genomic and no. 30. Int. J. Syst. Bacteriol. 39: 371. phenotypic analyses of avian Ureaplasma strains. Nippon Juigaku Zasshi 47: 901–909. Genus II. Ureaplasma 629

Hill, A.C. 1991a. Mycoplasma spermatophilum, a new species isolated newly recognized human pathogen Mycoplasma incognitus. Gene from human spermatozoa and cervix. Int. J. Syst. Bacteriol. 41: 93: 67–72. 229–233. Hum, S., A. Kessell, S. Djordjevic, R. Rheinberger, M. Hornitzky, W. Hill, A.C. 1991b. Mycoplasma oxoniensis, a new species isolated from chi- Forbes and J. Gonsalves. 2000. Mastitis, polyarthritis and abortion nese hamster conjunctivas. Int. J. Syst. Bacteriol. 41: 21–25. caused by Mycoplasma species bovine group 7 in dairy cattle. Aust. Hill, A.C. 1992. Mycoplasma simbae sp. nov., Mycoplasma leopharyngis sp. Vet. J. 78: 744–750. nov., and Mycoplasma leocaptivus sp. nov., isolated from lions. Int. J. Hyde, T., M. Gilbert, S. Schwartz, E. Zell, J. Watt, W. Thacker, D. Syst. Bacteriol. 42: 518–523. Talkington and R. Besser. 2001. Azithromycin prophylaxis during a Hill, A.C. 1993. Mycoplasma indiense sp. nov., isolated from the throats of hospital outbreak of Mycoplasma pneumoniae pneumonia. J. Infect. nonhuman primates. Int. J. Syst. Bacteriol. 43: 36–40. Dis. 183: 907–912. Hill, A., M. Tucker, D. Whittingham and I. Craft. 1987. Mycoplasmas Ivanics, E., R. Glávitis, G. Takacs, E. Molnár, Z. Bitay and M. Meder. and in vitro fertilization. Fertil. Steril. 47: 652–655. 1988. An outbreak of Mycoplasma anatis infection associated with ner- Hinz, K.H., H. Pfutzner and K.P. Behr. 1994. Isolation of mycoplasmas vous symptoms in large-scale duck flocks. Zentralbl. Veterinarmed. B from clinically healthy adult breeding geese in Germany. Zentralbl 35: 368–378. Veterinarmed B 41: 145–147. Jackson, G., E. Boughton and S.G. Hamer. 1981. An outbreak of Hirose, K., H. Kobayashi, N. Ito, Y. Kawasaki, M. Zako, K. Kotani, H. bovine mastitis associated with Mycoplasma canadense. Vet. Rec. 108: Ogawa and H. Sato. 2003. Isolation of Mycoplasmas from nasal swabs 31–32. of calves affected with respiratory diseases and antimicrobial suscep- Jacobs, E., A. Stuhlert, M. Drews, K. Pumpe, H. Schaefer, M. Kist and tibility of their isolates. J. Vet. Med. B Infect. Dis. Vet. Public Health W. Bredt. 1988. Host reactions to Mycoplasma pneumoniae infections 50: 347–351. in guinea-pigs preimmunized systemically with the adhesin of this Hodges, R.T., M. MacPherson, R.H. Leach and S. Moller. 1983. Isola- pathogen. Microb. Pathog. 5: 259–265. tion of Mycoplasma dispar from mastitis in dry cows. NZ Vet. J. 31: Jacobsson, B., I. Mattsby-Baltzer, B. Andersch, H. Bokstrom, R.M. Holst, 60–61. N. Nikolaitchouk, U.B. Wennerholm and H. Hagberg. 2003. Micro- Hoelzle, L. 2008. Haemotrophic mycoplasmas: recent advances in Myco- bial invasion and cytokine response in amniotic fluid in a Swedish plasma suis. Vet. Microbiol. 130: 215–226. population of women with preterm prelabor rupture of membranes. Hoffman, R.W., M.P. Luttrell, W.R. Davidson and D.H. Ley. 1997. Myco- Acta Obstet. Gynecol. Scand. 82: 423–431. plasmas in wild turkeys living in association with domestic fowl. J. Jain, N.C., D.E. Jasper and J.D. Dellinger. 1967. Cultural characteris- Wildl. Dis. 33: 526–535. tics and serological relationships of some mycoplasmas isolated from Hooper, P.T., L.A. Ireland and A. Carter. 1985. Mycoplasma polyarthritis in bovine sources. J. Gen. Microbiol. 49: 401–410. a cat with probable severe immune deficiency. Aust. Vet. J. 62: 352. Jan, G., M. Le Hénaff, C. Fontenelle and H. Wróblewski. 2001. Bio- Hopkins, P.M., D.S. Winlaw, P.N. Chhajed, J.L. Harkness, M.D. Horton, chemical and antigenic characterisation of Mycoplasma gallisepticum A.M. Keogh, M.A. Malouf and A.R. Glanville. 2002. Mycoplasma homi- membrane proteins P52 and P67 (pMGA). Arch. Microbiol. 177: nis infection in heart and lung transplantation. J. Heart Lung Trans- 81–90. plant 21: 1225–1229. Jasper, D.E., H. Erno, J.D. Dellinger and C. Christiansen. 1981. Myco- Horowitz, S., L. Duffy, B. Garrett, J. Stephens, J. Davis and G. Cassell. plasma californicum, a new species from cows. Int. J. Syst. Bacteriol. 1986. Can group- and serovar-specific proteins be detected in Ure- 31: 339–345. aplasma urealyticum? Pediatr. Infect. Dis. 5: S325–331. Jeansson, S. and J.E. Brorson. 1985. Elimination of mycoplasmas from Horowitz, S., J. Horowitz, D. Taylor-Robinson, S. Sukenik, R.N. Apte, J. cell cultures utilizing hyperimmune sera. Exp. Cell. Res. 161: 181– Bar-David, B. Thomas and C. Gilroy. 1994. Ureaplasma urealyticum in 188. Reiter’s syndrome. J. Rheumatol. 21: 877–882. Jensen, J.S. 2004. Mycoplasma genitalium: the aetiological agent of ure- Hoskins, J.D. 1991. Canine haemobartonellosis, canine hepatozoonosis, thritis and other sexually transmitted diseases. J. Eur. Acad. Derma- and feline cytauxzoonosis. Vet. Clin. North Am. Small Anim. Pract. tol. Venereol. 18: 1–11. 21: 129–140. Jensen, J.S., E. Bjornelius, B. Dohn and P. Lidbrink. 2004. Comparison Howard, C.J., D.H. Pocock and R.N. Gourlay. 1978. Base composition of first void urine and urogenital swab specimens for detection of of deoxyribonucleic acid from ureaplasmas isolated from various ani- Mycoplasma genitalium and Chlamydia trachomatis by polymerase chain mal species. Int. J. Syst. Bacteriol. 28: 599–601. reaction in patients attending a sexually transmitted disease clinic. Howard, C.J., R.N. Gourlay and S.G. Wyld. 1980. Isolation of a Virus, Sex. Transm. Dis. 31: 499–507. Mvbr1, from Mycoplasma-Bovirhinis. FEMS Microbiol. Lett. 7: 163– Johansson, K.E. and Pettersson B. 2002. Taxonomy of Mollicutes. In 165. Molecular biology and pathogenicity of mycoplasmas (edited by Howard, C.J., D.H. Pocock and R.N. Gourlay. 1981. Polyacrylamide gel Razin and Herrmann). Kluwer Academic, New York, pp. 1–30. electrophoretic comparison or the polypeptides from Ureaplasma Jones, G.E., A. Foggie, A. Sutherland and D.B. Harker. 1976. Mycoplas- species isolated from cattle and humans. Int. J. Syst. Bacteriol. 31: mas and ovine keratoconjunctivitis. Vet. Rec. 99: 137–141. 128–130. Jones, G.E. 1985. The pathogenicity of some ovine or caprine mycoplas- Howard, C.J. and R.N. Gourlay. 1982. Proposal for a second species mas in the lactating mammary gland of sheep and goats. J. Comp. within the genus Ureaplasma, Ureaplasma diversum sp. nov. Int. J. Syst. Pathol. 95: 305–318. Bacteriol. 32: 446–452. Jordan, F.T., J.N. Howse, M.P. Adams and O.O. Fatunmbi. 1981. The Howard, G.W. 1975. The experimental transmission of Eperythrozoon ovis isolation of Mycoplasma columbinum and M columborale from feral by mosquitoes. Parasitology 71: xxxiii. pigeons. Vet. Rec. 109: 450. Hsu, F.S., M.C. Liu, S.M. Chou, J.F. Zachary and A.R. Smith. 1992. Jordan, F.T.W. 1979. Avian mycoplasmas. In The Mycoplasmas, vol. 2 Evaluation of an enzyme-linked immunosorbent assay for detec- (edited by Tully Maniloff Whitcomb). Academic Press, New York, tion of Eperythrozoon suis antibodies in swine. Am. J. Vet. Res. 53: pp. 1–48. 352–354. Jordan, F.T.W., H. Ernø, G.S. Cottew, K.H. Kunz and L. Stipkovits. 1982. Hu, W.S., R.Y. Wang, R.S. Liou, J.W. Shih and S.C. Lo. 1990. Iden- Characterization and taxonomic description of five Mycoplasma sero- tification of an insertion-sequence-like genetic element in the vars (serotypes) of avian origin and their elevation to species rank 630 Family I. Mycoplasmataceae

and further evaluation of the taxonomic status of Mycoplasma syn- Klassen, T.P., N. Wiebe, K. Russell, K. Stevens, L. Hartling, W.R. oviae. Int. J. Syst. Bacteriol. 32: 108–115. Craig and D. Moher. 2002. Abstracts of randomized controlled Judicial Commission. 1958. Opinion 22. Status of the generic name trials presented at the society for pediatric research meeting: an Asterococcus and conservation of the generic name Mycoplasma. Int. example of publication bias. Arch. Pediatr. Adolesc. Med. 156: Bull. Bacteriol. Nomencl. Taxon. 8: 166–168. 474–479. Kakulphimp, J., L.R. Finch and J.A. Robertson. 1991. Genome sizes of Kleckner, A.L. 1960. Serotypes of avian pleuropneumonia-like organ- mammalian and avian Ureaplasmas. Int. J. Syst. Bacteriol. 41: 326– isms. Am. J. Vet. Res. 21: 274–280. 327. Kleven, S.H., C.S. Eidson and O.J. Fletcher. 1978. Airsacculitis induced Kaya, S., O. Poyraz, G. Gokce, H. Kilicarslan, K. Kaya and S. Ayan. 2003. in broilers with a combination of Mycoplasma gallinarum and respira- Role of genital mycoplasmata and other bacteria in urolithiasis. tory viruses. Avian Dis. 22: 707–716. Scand. J. Infect. Dis. 35: 315–317. Kleven, S.H. 1998. Mycoplasmas in the etiology of multifactorial respi- Keane, F.E., B.J. Thomas, C.B. Gilroy, A. Renton and D. Taylor-­Robinson. ratory disease. Poult. Sci. 77: 1146–1149. 2000. The association of Mycoplasma hominis, Ureaplasma urealyticum Kleven, S.H. 2003. Mycoplasmosis: other mycoplasmal infections. In and Mycoplasma genitalium with bacterial vaginosis: observations on Diseases of Poultry, 11th edn (edited by Saif and Barnes). Wiley- heterosexual women and their male partners. Int. J. STD AIDS 11: ­Blackwell, Hoboken, NJ. 356–360. Kleven, S.H. 2008. Control of avian Mycoplasma infections in commer- Kempf, I., F. Gesbert, E. Guinebert, G. M. and G. Bennejean. 1991. cial poultry. Avian Dis. 52: 367–374. Isolement et caractérisation d’une souche mycoplasmique chez Knox, C., P. Giffard and P. Timms. 1998. The phylogeny of Ureaplasma des faisans d’élevage. Recuil de Medecine Veterinaire 167: 1133– urealyticum based on the mba gene fragment. Int. J. Syst. Bacteriol. 1139. 48 Pt 4: 1323–1331. Kempf, I. 1998. DNA amplification methods for diagnosis and epide- Knox, C. and P. Timms. 1998. Comparison of PCR, nested PCR, and miological investigations of avian mycoplasmosis. Avian Pathol. 27: random amplified polymorphic DNA PCR for detection and typing 7–14. of Ureaplasma urealyticum in specimens from pregnant women. J. Clin. Kempf, I., C. Chastel, S. Ferris, F. Dufour-Gesbert, K.E. Johansson, B. Microbiol. 36: 3032–3039. Pettersson and A. Blanchard. 2000. Isolation of Mycoplasma columbo- Knox, C., J. Allan, J. Allan, W. Edirisinghe, D. Stenzel, F. Lawrence, D. rale from a fly (Musca domestica). Vet. Rec. 147: 304–305. Purdie and P. Timms. 2003. Ureaplasma parvum and Ureaplasma ure- Kenny, G. 1983. Inhibition of the growth of Ureaplasma urealyticum by a alyticum are detected in semen after washing before assisted repro- new urease inhibitor, flurofamide. Yale J. Biol. Med. 56: 717–722. ductive technology procedures. Fertil. Steril. 80: 921–929. Keymer, I.F., R.H. Leach, R.A. Clarke, M.E. Bardsley and R.R. McIntyre. Kobayashi, H., M. Runge, R. Schmidt, M. Kubo, K. Yamamoto and 1984. Isolation of Mycoplasma spp. from racing pigeons (Columba H. Kirchhoff. 1997. Mycoplasma lagogenitalium sp. nov., from the livia). Avian Pathol. 13: 65–74. preputial smegma of Afghan pikas (Ochotona rufescens rufescens). Int. Kidanemariam, A., J. Gouws, M. van Vuuren and B. Gummow. 2005. J. Syst. Bacteriol. 47: 1208–1211. Ulcerative balanitis and vulvitis of Dorper sheep in South Africa: Kokotovic, B., N.F. Friis and P. Ahrens. 2007. Mycoplasma alkalescens a study on its aetiology and clinical features. JS Afr. Vet. Assoc. 76: demonstrated in bronchoalveolar lavage of cattle in Denmark. Acta 197–203. Vet. Scand. 49: 2. Kikuth, W. 1928. Über Einen neuen Anämeerreger; Bartonella canis nov. Kong, F., G. James, Z. Ma, S. Gordon, W. Bin and G. Gilbert. 1999a. spec. Klin. Wochenschr. 7: 1729–1730. Phylogenetic analysis of Ureaplasma urealyticum–support for the estab- Kilian, M., M.B. Brown, T.A. Brown, E.A. Freundt and G.H. Cassell. lishment of a new species, Ureaplasma parvum. Int. J. Syst. Bacteriol. 1984. Immunoglobulin A1 protease activity in strains of Ureaplasma 49 Pt 4: 1879–1889. urealyticum. Acta Pathol. Microbiol. Immunol. Scand. [B]. 92: 61–64. Kong, F., X. Zhu, W. Wang, X. Zhou, S. Gordon and G.L. Gilbert. King, K.W. and K. Dybvig. 1994. Mycoplasmal cloning vectors derived 1999b. Comparative analysis and serovar-specific identification of from plasmid pKMK1. Plasmid 31: 49–59. ­multiple-banded antigen genes of Ureaplasma urealyticum biovar 1. J. Kirchhoff, H. 1978. Mycoplasma equigenitalium, a new species from cervix Clin. Microbiol. 37: 538–543. region of mares. Int. J. Syst. Bacteriol. 28: 496–502. Kong, F., Z. Ma, G. James, S. Gordon and G. Gilbert. 2000. Molecular Kirchhoff, H., P. Beyene, M. Fischer, J. Flossdorf, J. Heitmann, B. genotyping of human Ureaplasma species based on multiple-banded ­Khattab, D. Lopatta, R. Rosengarten, G. Seidel and C. Yousef. 1987. antigen (MBA) gene sequences. Int. J. Syst. Evol. Microbiol. 50 Pt 5: Mycoplasma mobile sp. nov., a new species from fish. Int. J. Syst. Bacte- 1921–1929. riol. 37: 192–197. Königsson, M.H., B. Pettersson and K.E. Johansson. 2001. Phylog- Kirchhoff, H., A. Binder, B. Liess, K.T. Friedhoff, J. Pohlenz, M. Stede eny of the seal mycoplasmas Mycoplasma phocae corrig., Mycoplasma and T. Willhaus. 1989. Isolation of mycoplasmas from diseased seals. phocicerebrale corrig. and Mycoplasma phocirhinis corrig. based on Vet. Rec. 124: 513–514. sequence analysis of 16S rDNA. Int. J. Syst. Evol. Microbiol. 51: Kirchhoff, H., R. Schmidt, H. Lehmann, H.W. Clark and A.C. Hill. 1389–1393. 1996. Mycoplasma elephantis sp. nov., a new species from elephants. Koshimizu, K., M. Ito, T. Magaribuchi and H. Kotani. 1983. Selective Int. J. Syst. Bacteriol. 46: 437–441. medium for isolation of ureaplasmas from animals. Nippon Juigaku Kirchhoff, H., K. Mohan, R. Schmidt, M. Runge, D.R. Brown, M.B. Zasshi 45: 263–268. Brown, C.M. Foggin, P. Muvavarirwa, H. Lehmann and J. Flossdorf. Koshimizu, K., R. Harasawa, I.J. Pan, H. Kotani, M. Ogata, E.B. ­Stephens 1997. Mycoplasma crocodyli sp. nov., a new species from crocodiles. Int. and M.F. Barile. 1987. Ureaplasma gallorale sp. nov. from the orophar- J. Syst. Bacteriol. 47: 742–746. ynx of chickens. Int. J. Syst. Bacteriol. 37: 333–338. Kirchoff, H. 1974. Neue spezies der Fam. Acholeplasmataceae und der Krause, D.C. and D. Taylor-Robinson. 1992. Mycoplasmas which infect Fam. Mykoplasmataceae bei Pferden. Zentralbl. Veterinarmed. B 21: humans. In Mycoplasmas: Molecular Biology and Pathogenesis 207–210. (edited by Maniloff, McElhaney, Finch and Baseman). American Kisary, J., A. El-Ebeedy and L. Stipkovits. 1976. Mycoplasma infection of Society for Microbiology, Washington, D.C., pp. 417–444. geese. II. Studies on pathogenicity of mycoplasmas in goslings and Krause, D.C. and M.F. Balish. 2004. Cellular engineering in a minimal goose and chicken embryos. Avian Pathol. 5: 15–20. microbe: structure and assembly of the terminal organelle of Myco- plasma pneumoniae. Mol. Microbiol. 51: 917–924. Genus II. Ureaplasma 631

Kreier, J.P. and M. Ristic. 1963. Morphologic, antigenic, and pathogenic Lemcke, R.M. 1979. Equine Mycoplasmas. In The Mycoplasmas Volume characteristics of Eperythrozoon ovis and Eperythrozoon wenyoni. Am. J. 2: Human and Animal Mycoplasmas, vol. 2 (edited by Tully and Whit- Vet. Res. 24: 488–500. comb). Academic Press, New York, pp. 177–189. Kreier, J.P. and M. Ristic. 1968. Haemobartonellosis, eperythrozoonosis, Lemcke, R.M. and H. Kirchhoff. 1979. Mycoplasma subdolum, a new spe- grahamellosis and ehrlichiosis. In Infectious Blood Diseases of Man cies isolated from horses. Int. J. Syst. Bacteriol. 29: 42–50. and Animals (edited by Weinman and Ristic). Academic Press, New Lemcke, R.M. and J. Poland. 1980. Mycoplasma fastidiosum: new species York, pp. 387–472. from horses. Int. J. Syst. Bacteriol. 30: 151–162. Kreier, J.P. and M. Ristic. 1984. Genus III. Haemobartonella; Genus IV. Eperyth- Lesnoff, M., G. Laval, P. Bonnet, S. Abdicho, A. Workalemahu, D. Kifle, rozoon. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by A. Peyraud, R. Lancelot and F. Thiaucourt. 2004. Within-herd spread Krieg and Holt). Williams & Wilkins, Baltimore, pp. 724–729. of contagious bovine pleuropneumonia in Ethiopian highlands. Prev Kusiluka, L.J., B. Ojeniyi, N.F. Friis, R.R. Kazwala and B. Kokotovic. Vet Med 64: 27–40. 2000. Mycoplasmas isolated from the respiratory tract of cattle and Levisohn, S. and S.H. Kleven. 2000. Avian mycoplasmosis (Mycoplasma goats in Tanzania. Acta Vet. Scand. 41: 299–309. gallisepticum). Rev. Sci. Tech. 19: 425–442. Lam, K., A. DaMassa and G. Ghazikhanian. 2004. Mycoplasma meleagridis- Ley, D.H., J.E. Berkhoff and J.M. McLaren. 1996. Mycoplasma gallisepti- induced lesions in the tarsometatarsal joints of turkey embryos. Avian cum isolated from house finches (Carpodacus mexicanus) with con- Dis. 48: 505–511. junctivitis. Avian Dis. 40: 480–483. Lamm, C.G., L. Munson, M.C. Thurmond, B.C. Barr and L.W. George. Ley, D.H., S.J. Geary, J.E. Berkhoff, J.M. McLaren and S. Levisohn. 2004. Mycoplasma otitis in California calves. J. Vet. Diagn. Invest. 1998. Mycoplasma sturni from blue jays and northern mockingbirds 16: 397–402. with conjunctivitis in Florida. J. Wildl. Dis. 34: 403–406. Lamster, I., J. Grbic, R. Bucklan, D. Mitchell-Lewis, H. Reynolds and Li, X., X. Jia, D. Shi, Y. Xiao, S. Hu, M. Liu, Z. Yuan and D. Bi. 2008. J. Zambon. 1997. Epidemiology and diagnosis of HIV-associated peri- Continuous in vitro Cultivation of Mycoplasma suis. Acta Veterinaria et odontal diseases. Oral Dis. 3 Suppl 1: S141–148. Zootechnica Sinica 38: 1142–1146. Langford, E.V. and R.H. Leach. 1973. Characterization of a Mycoplasma Li, Y., A. Brauner, B. Jonsson, I. van der Ploeg, O. Söder, M. Holst, J. isolated from infectious bovine keratoconjunctivitis: M. bovoculi sp. Jensen, H. Lagercrantz and K. Tullus. 2000. Ureaplasma urealyticum- nov. Can. J. Microbiol. 19: 1435–1444. induced production of proinflammatory cytokines by macrophages. Langford, E.V., H.L. Ruhnke and O. Onoviran. 1976. Mycoplasma Pediatr. Res. 48: 114–119. canadense, a new bovine species. Int. J. Syst. Bacteriol. 26: 212– Lierz, M., R. Schmidt, L. Brunnberg and M. Runge. 2000. Isolation of 219. Mycoplasma meleagridis from free-ranging birds of prey in Germany. J. Lartigue, C., J.I. Glass, N. Alperovich, R. Pieper, P.P. Parmar, C.A. Vet. Med. B Infect. Dis. Vet. Public Health 47: 63–67. Hutchison, 3rd, H.O. Smith and J.C. Venter. 2007. Genome trans- Lierz, M., R. Schmidt and M. Runge. 2002. Mycoplasma species isolated plantation in bacteria: changing one species to another. Science 317: from falcons in the Middle East. Vet. Rec. 151: 92–93. 632–638. Lierz, M., S. Deppenmeier, A. Gruber, S. Brokat and H. Hafez. 2007a. Lartigue, C., S. Vashee, M.A. Algire, R.Y. Chuang, G.A. Benders, L. Pathogenicity of Mycoplasma lipofaciens strain ML64 for turkey Ma, V.N. Noskov, E.A. Denisova, D.G. Gibson, N. Assad-Garcia, N. embryos. Avian Pathol. 36: 389–393. Alperovich, D.W. Thomas, C. Merryman, C.A. Hutchison, 3rd, H.O. Lierz, M., N. Hagen, N. Harcourt-Brown, S.J. Hernandez-Divers, D. Smith, J.C. Venter and J.I. Glass. 2009. Creating bacterial strains from Luschow and H.M. Hafez. 2007b. Prevalence of mycoplasmas in eggs genomes that have been cloned and engineered in yeast. Science from birds of prey using culture and a genus-specific Mycoplasma 325: 1693–1696. polymerase chain reaction. Avian Pathol. 36: 145–150. Lavricˇ, M., D. Bencˇina and M. Narat. 2005. Mycoplasma gallisepticum Lierz, M., R. Stark, S. Brokat and H. Hafez. 2007c. Pathogenicity of hemagglutinin V1hA, pyruvate dehydrogenase PdhA, lactate dehy- Mycoplasma lipofaciens strain ML64, isolated from an egg of a North- drogenase, and elongation factor Tu share epitopes with Mycoplasma ern Goshawk (Accipiter gentilis), for chicken embryos. Avian Pathol. imitans homologues. Avian Dis. 49: 507–513. 36: 151–153. Leach, R.H. 1967. Comparative studies of Mycoplasma of bovine origin. Lierz, M., N. Hagen, S.J. Hernadez-Divers and H.M. Hafez. 2008a. Ann. N. Y. Acad. Sci. 143: 305–316. Occurrence of mycoplasmas in free-ranging birds of prey in Ger- Leach, R.H. 1973. Further studies on classification of bovine strains of many. J. Wildl. Dis. 44: 845–850. Mycoplasmatales, with proposals for new species, Acholeplasma modicum Lierz, M., A. Jansen and H. Hafez. 2008b. Avian Mycoplasma lipofaciens and Mycoplasma alkalescens. J. Gen. Microbiol. 75: 135–153. transmission to veterinarian. Emerg. Infect. Dis. 14: 1161–1163. Leach, R.H. 1983. Preservation of Mycoplasma cultures and culture col- Lierz, M., E. Obon, B. Schink, F. Carbonell and H.M. Hafez. 2008c. The lections. In Methods in Mycoplasmology, vol. 1 (edited by Razin and role of mycoplasmas in a conservation project of the lesser kestrel Tully). Academic Press, New York, pp. 197–204. (Falco naumanni). Avian Dis. 52: 641–645. Leach, R.H., H. Ernø and K.J. MacOwan. 1993. Proposal for designa- Lin, J., M. Kendrick and E. Kass. 1972. Serologic typing of human geni- tion of F38-type caprine mycoplasmas as Mycoplasma capricolum subsp. tal T-mycoplasmas by a complement-dependent mycoplasmacidal capripneumoniae and consequent obligatory relegation of strains cur- test. J. Infect. Dis. 126: 658–663. rently classified as Mycoplasma capricolum (Tully, Barile, Edward, Theo­ Livingston, C.J. and B. Gauer. 1974. Serologic typing of T-strain Myco- dore, and Ernø 1974) to an additional new subspecies, M. capricolum plasma isolated from the respiratory and reproductive tracts of cattle subsp. capricolum subsp. nov. Int. J. Syst. Bacteriol. 43: 603–605. in the United States. Am. J. Vet. Res. 35: 1469–1471. Lee, G.Y. and G.E. Kenny. 1987. Humoral immune response to poly- Lo, S.C., M.M. Hayes, J.G. Tully, R.Y. Wang, H. Kotani, P.F. Pierce, peptides of Ureaplasma urealyticum in women with postpartum fever. D.L. Rose and J.W. Shih. 1992. Mycoplasma penetrans sp. nov., from J. Clin. Microbiol. 25: 1841–1844. the urogenital tract of patients with AIDS. Int. J. Syst. Bacteriol. 42: Lehmer, R.R., B.S. Andrews, J.A. Robertson, E.E. Stanbridge, L. de la 357–364. Maza and G.J. Friow. 1991. Polyarthiritis due to Ureaplasma urealyti- Lobo, E., M.C. García, H. Moscoso, S. Martínez and S.H. Kleven. 2004. cum infection in a patient with common variable immunodeficiency Strain heterogeneity in Mycoplasma pullorum isolates identified by (CVID): Similarities to rheumatoid arthritis. Ann. Rheum. Dis. 50: random amplified polymorphic DNA techniques. Spanish Journal of 574–576. Agricultural Research 2: 500–503. 632 Family I. Mycoplasmataceae

Lorenzon, S., L. Manso-Silvan and F. Thiaucourt. 2008. Specific real- Masover, G.K. and F.A. Becker. 1996. Detection of mycoplasmas by DNA time PCR assays for the detection and quantification of Mycoplasma staining and fluorescent antibody methodology. In Molecular and mycoides subsp. mycoides SC and Mycoplasma capricolum subsp. caprip- Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully neumoniae. Mol. Cell. Probes. 22: 324–328. and Razin). Academic Press, San Diego, pp. 419–429. Luo, W., H. Yu, Z. Cao, T.R. Schoeb, M. Marron and K. Dybvig. 2008. Matlow, A., C. Th’ng, D. Kovach, P. Quinn, M. Dunn and E. Wang. Association of Mycoplasma arthritidis mitogen with lethal toxicity but 1998. Susceptibilities of neonatal respiratory isolates of Ureaplasma not with arthritis in mice. Infect. Immun. 76: 4989–4998. urealyticum to antimicrobial agents. Antimicrob. Agents Chemother. Luttrell, M.P., T.H. Eleazer and S.H. Kleven. 1992. Mycoplasma gallopavo- 42: 1290–1292. nis in eastern wild turkeys. J. Wildl. Dis. 28: 288–291. Mattila, P.S., P. Carlson, A. Sivonen, J. Savola, R. Luosto, J. Salo and M. Lysnyansky, I., M. Calcutt, I. Ben-Barak, Y. Ron, S. Levisohn, B. Methé Valtonen. 1999. Life-threatening Mycoplasma hominis mediastinitis. and D. Yogev. 2009. Molecular characterization of newly identified Clin. Infect. Dis. 29: 1529–1537. IS3, IS4 and IS30 insertion sequence-like elements in Mycoplasma Mayer, D., M.P. Degiorgis, W. Meier, J. Nicolet and M. Giacometti. 1997. bovis and their possible roles in genome plasticity. FEMS Microbiol. Lesions associated with infectious keratoconjunctivitis in alpine ibex. Lett. 294: 172–182. J. Wildl. Dis. 33: 413–419. MacKenzie, C., B. Henrich and U. Hadding. 1996. Biovar-specific May, M., G.J. Ortiz, L.D. Wendland, D.S. Rotstein, R.F. Relich, M.F. Bal- epitopes of the urease enzyme of Ureaplasma urealyticum. J. Med. ish and D.R. Brown. 2007. Mycoplasma insons sp. nov., a twisted Myco- Microbiol. 45: 366–371. plasma from green iguanas (Iguana iguana). FEMS Microbiol. Lett. MacOwan, K.J., H.G. Jones, C.J. Randall and F.T. Jordan. 1981. Myco- 274: 298–303. plasma columborale in a respiratory condition of pigeons and experi- Mayer, D., M.P. Degiorgis, W. Meier, J. Nocolet and M. Giacometti. 1997. mental air sacculitis of chickens. Vet. Rec. 109: 562. Lesions associated with infectious keratoconjuctivitis in alpine ibex. Madden, D.L., K.E. Moats, W.T. London, E.B. Matthew and J.L. Sever. J. Widl. Dis. 33: 413–419. 1974. Mycoplasma moatsii, a new species isolated from recently Mayer, M. 1921. Über einige bakterienähnliche Parasiten der Erythrozyten imported Grivit monkeys (Cercopithecus aethiops). Int. J. Syst. Bacte- bei Menschen und Tieren. Arch. Schiffs Trop. Hyg. 25: 150–152. riol. 24: 459–464. Mazzali, R. and D. Taylor-Robinson. 1971. The behaviour of T-mycoplasmas Maeda, T., T. Shibahara, K. Kimura, Y. Wada, K. Sato, Y. Imada, in tissue culture. J. Med. Microbiol. 4: 125–138. Y. Ishikawa and K. Kadota. 2003. Mycoplasma bovis-associated suppura- McAuliffe, L., R. Ellis, K. Miles, R. Ayling and R. Nicholas. 2006. Biofilm tive otitis media and pneumonia in bull calves. J. Comp. Pathol. 129: formation by Mycoplasma species and its role in environmental persis- 100–110. tence and survival. Microbiology 152: 913–922. Manchee, R. and D. Taylor-Robinson. 1969. Enhanced growth of T-strain McAuliffe, L., R.D. Ayling, R.J. Ellis and R.A. Nicholas. 2008. Biofilm- mycoplasmas with N-2-hydroxyethylpiperazone-N¢-2-ethanesulfonic grown Mycoplasma mycoides subsp. mycoides SC exhibit both pheno- acid buffer. J. Bacteriol. 100: 78–85. typic and genotypic variation compared with planktonic cells. Vet. Manchee, R. and D. Taylor-Robinson. 1970. Lysis and protection of Microbiol. 129: 315–324. erythrocytes by T-mycoplasmas. J. Med. Microbiol. 3: 539–546. McElhaney, R.N. 1992a. Lipid composition, biosynthesis, and metabo- Manhart, L.E., S.M. Dutro, K.K. Holmes, C.E. Stevens, C.W. Critchlow, lism. In Mycoplasmas: Molecular Biology and Pathogenesis (edited D.A. Eschenbach and P.A. Totten. 2001. Mycoplasma genitalium is by Maniloff, McElhaney, Finch and Baseman). American Society for associated with mucopurulent cervicitis. Int. J. STD AIDS 12 Microbiology, Washington, D.C., pp. 231–258. (suppl. 2): 69. McElhaney, R.N. 1992b. Membrance function. In Mycoplasmas: Molec- Manso-Silván, L., E.M. Vilei, K. Sachse, S.P. Djordjevic, F. Thiaucourt ular Biology and Pathogenesis (edited by Maniloff, McElhaney, and J. Frey. 2009. Mycoplasma leachii sp. nov. as a new species designa- Finch and Baseman). American Society for Microbiology, Washing- tion for Mycoplasma sp. bovine group 7 of Leach, and reclassification ton, D.C., pp. 259–287. of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma McElhaney, R.N. 1992c. Membrane structure. In Mycoplasmas: Molecu- mycoides subsp. capri. Int. J. Syst. Evol. Microbiol. 59: 1353–1358. lar Biology and Pathogenesis (edited by Maniloff, McElhaney, Finch March, J.B., C. Gammack and R. Nicholas. 2000. Rapid detection of and Baseman). American Society for Microbiology, Washington, contagious caprine pleuropneumonia using a Mycoplasma capri- D.C., pp. 113–155. colum subsp. capripneumoniae capsular polysaccharide-specific McGarrity, G.J., D.L. Rose, V. Kwiatkowski, A.S. Dion, D.M. Phillips and antigen detection latex agglutination test. J. Clin. Microbiol. 38: J.G. Tully. 1983. Mycoplasma muris, a new species from laboratory 4152–4159. mice. Int. J. Syst. Bacteriol. 33: 350–355. Maré, C.J. and W.P. Switzer. 1965. Mycoplasm hyopneumoniae, a causative McLaughlin, B.G., P.S. McLaughlin and C.N. Evans. 1991. An Eperythro- agent of virus pig pneumonia. Vet. Med. 60: 841–845. zoon-like parasite of llamas: attempted transmission to swine, sheep, Markham, P.F., M.F. Duffy, M.D. Glew and G.F. Browning. 1999. A gene and cats. J. Vet. Diagn. Invest. 3: 352–353. family of Mycoplasma imitans closely related to the pMGA family of McMartin, D.A., K.J. MacOwan and L.L. Swift. 1980. A century of classi- Mycoplasma gallisepticum. Microbiology 145: 2095–2103. cal contagious caprine pleuropneumonia: from original description Marois, C., F. Dufour-Gesbert and I. Kempf. 2001. Comparison of to aetiology. Br. Vet. J. 136: 507–515. pulsed-field gel electrophoresis with random amplified polymor- Meloni, G.A., G. Bertoloni, F. Busolo and L. Conventi. 1980. Colony phic DNA for typing of Mycoplasma synoviae. Vet. Microbiol. 79: 1–9. morphology, ultrastructure and morphogenesis in Mycoplasma homi- Marois, C., J. Le Carrou, M. Kobisch and A.V. Gautier-Bouchardon. nis, Acholeplasma laidlawii and Ureaplasma urealyticum. J. Gen. Micro- 2007. Isolation of Mycoplasma hyopneumoniae from different sampling biol. 116: 435–443. sites in experimentally infected and contact SPF piglets. Vet. Micro- Messick, J.B., P.G. Walker, W. Raphael, L. Berent and X. Shi. 2002. biol. 120: 96–104. ‘Candidatus Mycoplasma haemodidelphidis’ sp. nov., ‘Candidatus Mason, R.W. and P. Statham. 1991. The determination of the level of Mycoplasma haemolamae’ sp. nov. and Mycoplasma haemocanis comb. Eperythrozoon ovis parasitaemia in chronically infected sheep and its nov., haemotrophic parasites from a naturally infected opossum significance to the spread of infection. Aust. Vet. J. 68: 115–116. (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis famil- Masover, G., S. Razin and L. Hayflick. 1977. Localization of enzymes iaris): phylogenetic and secondary structural relatedness of their in Ureaplasma urealyticum (T-strain mycoplasma). J. Bacteriol. 130: 16S rRNA genes to other mycoplasmas. Int. J. Syst. Evol. Microbiol. 297–302. 52: 693–698. Genus II. Ureaplasma 633

Messick, J.B. 2003. New perspectives about Hemotrophic Mycoplasma Muhlrad, A., I. Peleg, J. Robertson, I. Robinson and I. Kahane. 1981. (formerly, Haemobartonella and Eperythrozoon species) infections in Acetate kinase activity in mycoplasmas. J. Bacteriol. 147: 271–273. dogs and cats. Vet. Clin. N. Am. Small Anim. Pract. 33: 1453–1465. Murakami, S., M. Miyama, A. Ogawa, J. Shimada and T. Nakane. 2002. Meyer, R. and W. Clough. 1993. Extragenital Mycoplasma hominis infec- Occurrence of conjunctivitis, sinusitis and upper region tracheitis tions in adults: emphasis on immunosuppression. Clin. Infect. Dis. in Japanese quail (Coturnix coturnix japonica), possibly caused by 17 Suppl 1: S243–249. Mycoplasma gallisepticum accompanied by Cryptosporidium sp. infec- Meyling, A. and N.F. Friis. 1972. Serological identification of a new por- tion. Avian Pathol. 31: 363–370. cine Mycoplasma species, M. flocculare. Acta Vet. Scand. 13: 287–289. Nagata, K., E. Takagi, H. Satoh, H. Okamura and T. Tamura. 1995. Michel, J.C., B. de Thoisy and H. Contamin. 2000. Chemotherapy of Growth inhibition of Ureaplasma urealyticum by the proton pump haemobartonellosis in squirrel monkeys (Saimiri sciureus). J. Med. inhibitor lansoprazole: direct attribution to inhibition by lansopra- Primatol. 29: 85–87. zole of urease activity and urea-induced ATP synthesis in U. urealyti- Mikaelian, I., D.H. Ley, R. Claveau, M. Lemieux and J.P. Berube. 2001. cum. Antimicrob. Agents Chemother. 39: 2187–2192. Mycoplasmosis in evening and pine grosbeaks with conjunctivitis in Nagatomo, H., H. Kato, T. Shimizu and B. Katayama. 1997. Isolation of Quebec. J. Wildl. Dis. 37: 826–830. mycoplasmas from fantail pigeons. J. Vet. Med. Sci. 59: 461–462. Miles, R.J. 1992a. Catabolism in Mollicutes. J. Gen. Microbiol. 138: 1773– Nakane, D. and M. Miyata. 2007. Cytoskeletal “jellyfish” structure of 1783. Mycoplasma mobile. Proc. Natl. Acad. Sci. USA 104: 19518–19523. Miles, R.J. 1992b. Cell nutrition and growth. In Mycoplasmas: Molecu- Nakane, D. and M. Miyata. 2009. Cytoskeletal asymmetrical dumbbell lar Biology and Pathogenesis (edited by Maniloff, McElhaney, Finch structure of a gliding mycoplasma, Mycoplasma gallisepticum, revealed and Baseman). American Society for Microbiology, Washington, by negative-staining electron microscopy. J. Bacteriol. 191: 3256– D.C., pp. 23–40. 3264. Mirsalimi, S., S. Rosendal and R. Julian. 1989. Colonization of the intes- Naot, Y., J.G. Tully and H. Ginsburg. 1977. Lymphocyte activation by tine of turkey embryos exposed to Mycoplasma iowae. Avian Dis. 33: various Mycoplasma strains and species. Infect. Immun. 18: 310– 310–315. 317. Miyata, M., H. Yamamoto, T. Shimizu, A. Uenoyama, C. Citti and Neimark, H., D. Mitchelmore and R.H. Leach. 1998. An approach R. Rosengarten. 2000. Gliding mutants of Mycoplasma mobile: rela- to characterizing uncultivated prokaryotes the Grey Lung agent tionships between motility and cell morphology, cell adhesion and and proposal of a Candidatus taxon for the organism, ‘Candidatus microcolony formation. Microbiology 146: 1311–1320. Mycoplasma ravipulmonis’. Int. J. Syst. Bacteriol. 48: 389–394. Miyata, M., W.S. Ryu and H.C. Berg. 2002. Force and velocity of Myco- Neimark, H., K.E. Johansson, Y. Rikihisa and J.G. Tully. 2001. Proposal plasma mobile gliding. J. Bacteriol. 184: 1827–1831. to transfer some members of the genera Haemobartonella and Eperyth- Moalic, P.Y., I. Kempf, F. Gesbert and F. Laigret. 1997. Identification of rozoon to the genus Mycoplasma with descriptions of ‘Candidatus Myco- two pathogenic avian mycoplasmas as strains of Mycoplasma pullorum. plasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, ‘Candidatus Int. J. Syst. Bacteriol. 47: 171–174. Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyonii’. Int. J. Mohan, K., C.M. Foggin, P. Muvavarirwa and J. Honywill. 1997. Vaccina- Syst. Evol. Microbiol. 51: 891–899. tion of farmed crocodiles (Crocodylus niloticus) against Mycoplasma Neimark, H., A. Barnaud, P. Gounon, J.-C. Michel and H. Contamin. crocodyli infection. Vet. Rec. 141: 476. 2002a. The putative haemobartonella that influences Plasmodium Mohan, K., C.M. Foggin, F. Dziva and P. Muvavarirwa. 2001. Vaccination falciparum parasitaemia in squirrel monkeys is a haemotrophic myco- to control an outbreak of Mycoplasma crocodyli infection. Onderste- plasma. Microbes Infect. 4: 693–698. poort J. Vet. Res. 68: 149–150. Neimark, H., B. Hoff and M. Ganter. 2004. Mycoplasma ovis comb. nov. Moise, N.S., J.W. Crissman, J.F. Fairbrother and C. Baldwin. 1983. (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic Mycoplasma gateae arthritis and tenosynovitis in cats: case report anaemia in sheep and goats. Int. J. Syst. Evol. Microbiol. 54: 365– and experimental reproduction of the disease. Am. J. Vet. Res. 44: 371. 16–21. Neimark, H., W. Peters, B.L. Robinson and L.B. Stewart. 2005. Phylo- Monecke, S., J. Helbig and E. Jacobs. 2003. Phase variation of the multi- genetic analysis and description of Eperythrozoon coccoides, proposal ple banded protein in Ureaplasma urealyticum and Ureaplasma parvum. to transfer to the genus Mycoplasma as Mycoplasma coccoides comb. Int. J. Med. Microbiol. 293: 203–211. nov. and Request for an Opinion. Int. J. Syst. Evol. Microbiol. 55: Montagnier, L., A. Blanchard, D. Guetard, M. Lemaitre, A.M. Dirienzo, 1385–1391. S. Chamaret, Y. Henin, E. Bahraoui, C. Dauguet, C. Axler, M. Neimark, H.C., K.E. Johansson, Y. Rikihisa and J.G. Tully. 2002b. Revi- Kirstetter, R. Roue, G. pialoux and B. Dupont. 1990. A possible role sion of haemotrophic Mycoplasma species names. Int. J. Syst. Evol. of mycoplasmas as cofactors in AIDS. In Retroviruses of Human AIDS Microbiol. 52: 683. and Related Animal Diseases (edited by Girard and Valette). Col- Neitz, W.O., R.A. Alexander and P.J. de Toit. 1934. Eperythrozoon ovis (sp. loque de Cent Gardes, Foundation Merieux, Lyon, pp. 9–17. nov.) infection in sheep. Onderstepoort J. Vet. Sci. 3: 263–274. Montes, A., D. Wolfe, E. Welles, J. Tyler and E. Tepe. 1994. Infertility Nelson, J.B. and M.J. Lyons. 1965. Phase-contrast and electron micros- associated with Eperythrozoon wenyonii infection in a bull. J. Am. Vet. copy of murine strains of Mycoplasma. J. Bacteriol. 90: 1750–1763. Med. Assoc. 204: 261–263. Neyrolles, O., S. Ferris, N. Behbahani, L. Montagnier and A. Blanchard. Moss, T., C. Knox, S. Kallapur, I. Nitsos, C. Theodoropoulos, J. Newn- 1996. Organization of Ureaplasma urealyticum urease gene cluster and ham, M. Ikegami and A. Jobe. 2008. Experimental amniotic fluid expression in a suppressor strain of Escherichia coli. J. Bacteriol. 178: infection in sheep: effects of Ureaplasma parvum serovars 3 and 6 2725. on preterm or term fetal sheep. Am. J. Obstet. Gynecol. 198: 122. Niang, M., R.F. Rosenbusch, J.J. Andrews and M.L. Kaeberle. 1998. e121–128. Demonstration of a capsule on Mycoplasma ovipneumoniae. Am. J. Vet. Mouches, C., D. Taylor-Robinson, L. Stipkovits and J. Bove. 1981. Com- Res. 59: 557–562. parison of human and animal Ureaplasmas by one- and two-dimen- Nicholas, R.A., A. Greig, S.E. Baker, R.D. Ayling, M. Heldtander, K.-E. sional protein analysis on polyacrylamide slab gel. Ann. Microbiol. Johansson, B.M. Houshaymi and R.J. Miles. 1998. Isolation of Myco- (Paris) 132B: 171–196. plasma fermentans from a sheep. Vet. Rec. 142: 220–221. 634 Family I. Mycoplasmataceae

Nicholas, R.A., Y.C. Lin, K. Sachse, H. Hotzel, K. Parham, L. McAuliffe, Piot, P. 1977. Comparison of growth inhibition and immunofluores- R.J. Miles, D.P. Kelly and A.P. Wood. 2008. Proposal that the strains of cence tests in serotyping clinical isolates of Ureaplasma urealyticum. Br. the Mycoplasma ovine/caprine serogroup 11 be reclassified as Myco- J. Vener. Dis. 53: 186–189. plasma bovigenitalium. Int. J. Syst. Evol. Microbiol. 58: 308–312. Pitcher, D., M. Sillis and J.A. Robertson. 2001. Simple method for deter- Nikol’skii, S.N. and S.N. Slipchenko. 1969. Experiments in the trans- mining biovar and serovar types of Ureaplasma urealyticum clinical iso- mission of Eperythrozoon ovis by the ticks H. plumbeum and Rh. bursa. lates using PCR-single-strand conformation polymorphism analysis. Veterinariia (Russian) 5: 46. J. Clin. Microbiol. 39: 1840–1844. Nolan, P.M., S.R. Roberts and G.E. Hill. 2004. Effects of Mycoplasma gal- Pitcher, D., D. Windsor, H. Windsor, J. Brabbury, C. Yavari, J.S. Jensen, lisepticum on reproductive success in house finches. Avian Dis. 48: C. Ling and D. Webster. 2005. Mycoplasma amphoriforme sp. nov., a new 879–885. species isolated from a patient with chronic brochopneumonia. Int. Novy, M.J., L. L. Duffy, M.K. Axhelm, D.W. Sadowsky, S.S. Witkin, M.G. J. Syst. Evol. Microbiol. 55: 2589–2594. Gravett, Cassell, G.H. and K.B. Waites. 2009. Congenital and oppor- Pollack, J. 1986. Metabolic distinctiveness of ureaplasmas. Pediatr. tunistic infections: Ureaplasma species and Mycoplasma hominis. Repro- Infect. Dis. 5: S305–307. ductive Science 16: 56–70. Pollack, J.D. 1992. Carbohydrate metabolism and energy conserva- Nowak, J. 1929. Morphologie, nature et cycle évolutif du microbe de tion. In Mycoplasmas: Molecular Biology and Pathogenesis (edited la péripneumonie des bovidés. Ann. Inst. Pasteur (Paris) 43: 1330– by Maniloff, McElhaney, Finch and Baseman). American Society for 1352. Microbiology, Washington, D.C., pp. 181–200. Nunoya, T., T. Yagihashi, M. Tajima and Y. Nagasawa. 1995. Occurrence Pollack, J.D., M.V. Williams, J. Banzon, M.A. Jones, L. Harvey and of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum J.G. Tully. 1996. Comparative metabolism of Mesoplasma, Ento- in layer chickens. Vet. Pathol. 32: 11–18. moplasma, Mycoplasma, and Acholeplasma. Int. J. Syst. Bacteriol. 46: O’Brien, S.J., J. Simonson, S. Razin and M.F. Barile. 1983. On the distri- 885–890. bution and characteristics of isozyme expression in Mycoplasma. The Pollack, J.D. 1997. Mycoplasma genes: a case for reflective annotation. Yale Journal of Biology and Medicine 56: 701–708. Trends Microbiol. 5: 413–419. Oaks, J.L., S.L. Donahoe, F.R. Rurangirwa, B.A. Rideout, M. Gilbert and Pollack, J.D. 2001. Ureaplasma urealyticum: an opportunity for combina- M.Z. Virani. 2004. Identification of a novel Mycoplasma species from torial genomics. Trends Microbiol. 9: 169–175. an Oriental white-backed vulture (Gyps bengalensis). J. Clin. Micro- Pollack, J.D. 2002. The necessity of combining genomic and enzymatic biol. 42: 5909–5912. data to infer metabolic function and pathways in the smallest bacte- Olson, N.O., K.M. Kerr and A. Campbell. 1964. Control of infectious syno- ria: amino acid, purine and pyrimidine metabolism in mollicutes. vitis. 13. The antigen study of three strains. Avian Dis. 8: 209–214. Front. Biosci. 7: d1762–1781. Paessler, M., A. Levinson, J.B. Patel, M. Schuster, M. Minda and Poveda, J., J. Carranza, A. Miranda, A. Garrido, M. Hermoso, A. Fer- I. ­Nachamkin. 2002. Disseminated Mycoplasma orale infection in a nandez and J. Domenech. 1990. An epizootiological study of avian patient with common variable immunodeficiency syndrome. Diagn. mycoplasmas in southern Spain. Avian Pathol. 19: 627–633. Microbiol. Infect. Dis. 44: 201–204. Poveda, J.B., J. Giebel, J. Flossdorf, J. Meier and H. Kirchhoff. 1994. Panangala, V.S., J.S. Stringfellow, K. Dybvig, A. Woodard, F. Sun, D.L. Mycoplasma buteonis sp. nov. Mycoplasma falconis sp. nov. and Myco- Rose and M.M. Gresham. 1993. Mycoplasma corogypsi sp. nov., a new plasma gypis sp. nov. three species from birds of prey. Int. J. Syst. Bac- species from the footpad abscess of a black vulture, Coragyps atratus. teriol. 44: 94–98. Int. J. Syst. Bacteriol. 43: 585–590. Prullage, J.B., R.E. Williams and S.M. Gaafar. 1993. On the transmissibil- Papazisi, L., T. Gorton, G. Kutish, P. Markham, G. Browning, D. Nguyen, ity of Eperythrozoon suis by Stomoxys calcitrans and Aedes aegypti. Vet. S. Swartzell, A. Madan, G. Mahairas and S. Geary. 2003. The com- Parasitol. 50: 125–135. plete genome sequence of the avian pathogen Mycoplasma gallisepti- Purcell, R., D. Taylor-Robinson, D. Wong and R. Chanock. 1966. Color cum strain R(low). Microbiology 149: 2307–2316. test for the measurement of antibody to T-strain mycoplasmas. J. Pennycott, T., C. Dare, C. Yavari and J. Bradbury. 2005. Mycoplasma ­Bacteriol. 92: 6–12. sturni and Mycoplasma gallisepticum in wild birds in Scotland. Vet. Rec. Pye, G.W., D.R. Brown, M.F. Nogueira, K.A. Vliet, T.R. Schoeb, E.R. 156: 513–515. Jacobson and R.A. Bennett. 2001. Experimental inoculation of Pereyre, S., P. Gonzalez, B. de Barbeyrac, A. Darnige, H. Renaudin, A. broad-nosed caimans (Caiman latirostris) and Siamese crocodiles Charron, S. Raherison, C. Bébéar and C.M. Bébéar. 2002. Mutations (Crocodylus siamensis) with Mycoplasma alligatoris. J. Zoo Wildl. Med. in 23S rRNA account for intrinsic resistance to macrolides in Myco- 32: 196–201. plasma hominis and Mycoplasma fermentans and for acquired resistance Ramírez, A., C. Naylor, D. Pitcher and J. Bradbury. 2008. High inter- to macrolides in M. hominis. Antimicrob. Agents Chemother. 46: species and low intra-species variation in 16S-23S rDNA spacer 3142–3150. sequences of pathogenic avian mycoplasmas offers potential use as a Persson, A., B. Pettersson, G. Bölske and K.-E. Johansson. 1999. Diag- diagnostic tool. Vet. Microbiol. 128: 279–287. nosis of contagious bovine pleuropneumonia by PCR-laser- induced Raviv, Z. and S. Kleven. 2009. The development of diagnostic real-time fluorescence and PCR-restriction endonuclease analysis based on the TaqMan PCrs for the four pathogenic avian mycoplasmas. Avian Dis. 16S rRNA genes of Mycoplasma mycoides subsp. mycoides SC. J. Clin. 53: 103–107. Microbiol. 37: 3815–3821. Razin, S. 1979. Isolation and characterization of Mycoplasma mem- Peterson, J.E., A.W. Rodwell and E.S. Rodwell. 1973. Occurrence and branes. In The Mycoplasmas, vol. 1 (edited by Barile and Tully). Aca- ultrastructure of a variant (rho) form of Mycoplasma. J. Bacteriol. 115: demic Press, New York, pp. 213–229. 411–425. Razin, S. 1983. Cell lysis and isolation of membranes. In Methods in Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2000. Updated Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, phylogenetic description of the Mycoplasma hominis cluster (Weisburg New York, pp. 225–233. et al. 1989) based on 16S rDNA sequences. Int. J. Syst. Evol. Micro- Razin, S., D. Yogev and Y. Naot. 1998. Molecular biology and pathoge- biol. 50: 291–301. nicity of mycoplasmas. Microbiol. Mol. Biol. Rev. 62: 1094–1156. Pilo, P., B. Fleury, M. Marenda, J. Frey and E. Vilei. 2003. Prevalence Razin, S. and R. Herrmann (editors). 2002. Molecular Biology and and distribution of the insertion element ISMag1 in Mycoplasma aga- Pathogenicity of Mycoplasmas. Academic/Plenum Press, ­London. lactiae. Vet. Microbiol. 92: 37–48. Genus II. Ureaplasma 635

Reece, R.L., L. Ireland and P.C. Scott. 1986. Mycoplasmosis in racing Robertson, J.A. and G.W. Stemke. 1995. Measurement of Mollicute pigeons. Aust. Vet. J. 63: 166–167. Growth by ATP-Dependent Luminometry. In Molecular and Diag- Regendanz, P. and W. Kikuth. 1928. Über Aktivierung labiler Infek- nostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and tionen duch Entmilzung (Piroplasma canis, Nuttalia brasiliensis, Barto- Tully). Academic Press, San Diego, CA, pp. 65–71. nella opossum, Spirochaeta didelphydis). Arch. f. Schiffs. U. Tropenhyg. Rocha, E. and A. Blanchard. 2002. Genomic repeats, genome plasticity 32: 587–593. and the dynamics of Mycoplasma evolution. Nucleic Acids Res. 30: Relich, R.F., A.J. Friedberg and M.F. Balish. 2009. Novel cellular orga- 2031–2042. nization in a gliding Mycoplasma, Mycoplasma insons. J. Bacteriol. 191: Rodwell, A. and R.F. Whitcomb. 1983. Methods for direct and indirect 5312–5314. measurement of Mycoplasma growth. In Methods in Mycoplasmol- Reyes, L., M. Reinhard and M. Brown. 2009. Different inflammatory ogy, vol. 1 (edited by Razin and Tully). Academic Press, New York, responses are associated with Ureaplasma parvum-induced UTI and pp. 185–196. urolith formation. BMC Infect. Dis. 9: 9. Rodwell, A.W. 1983. Defined and partly defined media. In Methods in Roberts, D.H. 1964. The isolation of an influenza A virus and a Myco- Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, plasma associated with duck sinusitis. Vet. Rec. 76: 470–473. New York, pp. 163–172. Roberts, M. and G. Kenny. 1986. Dissemination of the tetM tetracycline Roifman, C., C. Rao, H. Lederman, S. Lavi, P. Quinn and E. Gelfand. resistance determinant to Ureaplasma urealyticum. Antimicrob. Agents 1986. Increased susceptibility to Mycoplasma infection in patients with Chemother. 29: 350–352. hypogammaglobulinemia. Am. J. Med. 80: 590–594. Robertson, J. and E. Smook. 1976. Cytochemical evidence of extramem- Romano, N., G. Tolone, F. Ajello and R. La Licata. 1980. Adenosine branous carbohydrates on Ureaplasma urealyticum (T-strain Myco- 5′-triphosphate synthesis induced by urea hydrolysis in Ureaplasma plasma). J. Bacteriol. 128: 658–660. urealyticum. J. Bacteriol. 144: 830–832. Robertson, J. 1978. Bromothymol blue broth: improved medium for Rose, D.L., J.G. Tully and E.V. Langford. 1978. Mycoplasma citelli, a detection of Ureaplasma urealyticum (T-strain mycoplasma). J. Clin. new species from ground squirrels. Int. J. Syst. Bacteriol. 28: 567– Microbiol. 7: 127–132. 572. Robertson, J. and G. Stemke. 1979. Modified metabolic inhibition test Rosenbusch, R.F. and F.C. Minion. 1992. Cell envelope: morphology for serotyping strains of Ureaplasma urealyticum (T-strain Mycoplasma). and biochemistry. In Mycoplasmas: Molecular Biology and Pathogen- J. Clin. Microbiol. 9: 673–676. esis (edited by Maniloff, McElhaney, Finch and Baseman). American Robertson, J., J. Coppola and O. Heisler. 1981. Standardized method Society for Microbiology, Washington, D.C., pp. 73–77. for determining antimicrobial susceptibility of strains of Ureaplasma Røsendal, S. 1974. Mycoplasma molare, a new canine Mycoplasma species. urealyticum and their response to tetracycline, erythromycin, and Int. J. Syst. Bacteriol. 24: 125–130. rosaramicin. Antimicrob. Agents Chemother. 20: 53–58. Røsendal, S. 1975. Canine mycoplasmas: serological studies of type and Robertson, J. 1982. Effect of gaseous conditions on isolation and growth reference strains, with a proposal for the new species, Mycoplasma of Ureaplasma urealyticum on agar. J. Clin. Microbiol. 15: 200–203. opalescens. Acta Pathol. Microbiol. Scand. Sect. B 83: 463–470. Robertson, J. and G. Stemke. 1982. Expanded serotyping scheme for Røsendal, S. and O. Vinther. 1977. Experimental mycoplasmal pneu- Ureaplasma urealyticum strains isolated from humans. J. Clin. Micro- monia in dogs: electron microscopy of infected tissue. Acta Pathol. biol. 15: 873–878. Microbiol. Scand. (B) 85B: 462–465. Robertson, J., M. Alfa and E. Boatman. 1983. Morphology of the cells Røsendal, S. 1979. Canine and feline mycoplasmas. In The Mycoplas- and colonies of Mycoplasma hominis. Sex Transm. Dis. 10: 232–239. mas (edited by Tully and Whitcomb). Academic Press, New York, Robertson, J. and M. Chen. 1984. Effects of manganese on the growth pp. 217–234. and morphology of Ureaplasma urealyticum. J. Clin. Microbiol. 19: Rosengarten, R., S. Levisohn and D. Yogev. 1995. A 41-kDa variable sur- 857–864. face protein of Mycoplasma gallisepticum has a counterpart in Myco- Robertson, J., M. Stemler and G. Stemke. 1984. Immunoglobulin A plasma imitans and Mycoplasma iowae. FEMS Microbiology Letters 132: protease activity of Ureaplasma urealyticum. J. Clin. Microbiol. 19: 115–123. 255–258. Röske, K., M. Calcutt and K. Wise. 2004. The Mycoplasma fermentans Robertson, J., G. Stemke, S. Maclellan and D. Taylor. 1988. Character- prophage phiMFV1: genome organization, mobility and variable ization of tetracycline-resistant strains of Ureaplasma urealyticum. J. expression of an encoded surface protein. Mol. Microbiol. 52: 1703– Antimicrob. Chemother. 21: 319–332. 1720. Robertson, J., A. Vekris, C. Bebear and G. Stemke. 1993. Polymerase Ross, R.F. and J.A. Karmon. 1970. Heterogeneity among strains of Myco- chain reaction using 16S rRNA gene sequences distinguishes the two plasma granularum and identification of Mycoplasma hyosynoviae, sp. n. biovars of Ureaplasma urealyticum. J. Clin. Microbiol. 31: 824–830. J. Bacteriol. 103: 707–713. Robertson, J.A., G. Stemke, J.J. Davis, R. Harasawa, D. Thirkell, F. Kong, Ross, R.F. 1992. Mycoplasmal diseases. In Diseases of Swine, 7th edn M. Shepard and D. Ford. 2002. Proposal of Ureaplasma parvum sp. (edited by Lemanske, Jr, Straw, Mengeling, D’Allaire and Taylor). nov. and emended description of Ureaplasma urealyticum (Shepard Iowa State University Press, Ames, IA, pp. 537–551. et al. 1974) Robertson et al. 2001. Int. J. Syst. Evol. Microbiol. 52: Ruhnke, H., N. Palmer, P. Doig and R. Miller. 1984. Bovine abortion 587–597. and neonatal death associated with Ureaplasma diversum. Theriog- Robertson, J.A., L.E. Pyle, G.W. Stemke and L.R. Finch. 1990. Human enology 21: 295–301. ureaplasmas show diverse genome sizes by pulsed-field electrophore- Ruhnke, H.L. and S. Madoff. 1992. Mycoplasma phocidae sp. nov., iso- sis. Nucleic Acids Res. 18: 1451–1455. lated from harbor seals (Phoca vitulina L.). Int. J. Syst. Bacteriol. 42: Robertson, J.A. and R. Sherburne. 1991. Hemadsorption by colonies of 211–214. Ureaplasma urealyticum. Infect. Immun. 59: 2203–2206. Ruifu, Y., Z. Minli, Z. Guo and X. Wang. 1997. Biovar diversity is Robertson, J.A., L.A. Howard, C.L. Zinner and G.W. Stemke. 1994. reflected by variations of genes encoding urease of Ureaplasma ure- Comparison of 16S rRNA genes within the T960 and parvo biovars alyticum. Microbiol. Immunol. 41: 625–627. of ureaplasmas isolated from humans. Int. J. Syst. Bacteriol. 44: Ruiter, M. and H.M. Wentholt. 1955. Isolation of a pleuropneumonia- 836–838. like organism from a skin lesion associated with a fusospirochetal flora. J. Invest. Dermatol. 24: 31–34. 636 Family I. Mycoplasmataceae

Sabin, A.B. 1941. The filterable microorganisms of the pleuropneumo- Shepard, M.C. 1983. Culture media for ureaplasmas. In Methods in nia group. Bacteriol. Rev. 5: 1–66. Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, Salih, M.M., N.F. Friis, S.N. Aarseculeratne, E.A. Freundt and C. Chris- New York, pp. 137–146. tiansen. 1983. Mycoplasma mustelae, a new species from mink. Int. Syst. Shepard, M.C. and J.A. Robertson. 1986. Calcium chloride as an indica- Bacteriol. 33: 476–479. tor for colonies of Ureaplasma urealyticum. The Pediatric Infectious Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, C. Diseases Journal 5: S349. Yoshino, A. Horino, T. Shiba, T. Sasaki and M. Hattori. 2002. The Shiferaw, G., S. Tariku, G. Ayelet and Z. Abebe. 2006. Contagious complete genomic sequence of Mycoplasma penetrans, an intracel- caprine pleuropneumonia and Mannheimia haemolytica-associated lular bacterial pathogen in humans. Nucleic Acids Res. 30: 5293– acute respiratory disease of goats and sheep in Afar Region, Ethiopia. 5300. Rev. Sci. Tech. 25: 1153–1163. Sayed, I. and G.E. Kenny. 1980. Comparison of the proteins and poly- Shimizu, T., H. Erno and H. Nagatomo. 1978. Isolation and char- peptides of the eight serotypes of Ureaplasma urealyticum by isoelectric acterization of Mycoplasma columbinum and Mycoplasma colum- focussing and sodium dodecyl sulfate-polyacrylamide gel electropho- borale, two new species from pigeons. Int. J. Syst. Bacteriol. 28: resis. Int. J. Syst. Bacteriol. 30: 33–41. 538–546. Scanziani, E., S. Paltrinieri, M. Boldini, V. Grieco, C. Monaci, A.M. Shimizu, T., K. Numano and K. Uchida. 1979. Isolation and identifica- Giusti and G. Mandelli. 1997. Histological and immunohistochemi- tion of mycoplasmas from various birds: an ecological study. Jpn. J. cal findings in thoracic lymph nodes of cattle with contagious bovine Vet. Sci. 41: 273–282. pleuropneumonia. J. Comp. Pathol. 117: 127–136. Shobokshi, A. and M. Shaarawy. 2002. Maternal serum and amniotic Schelonka, R., B. Katz, K. Waites and D.J. Benjamin. 2005. Critical fluid cytokines in patients with preterm premature rupture of mem- appraisal of the role of Ureaplasma in the development of bronchopul- branes with and without intrauterine infection. Int. J. Gynaecol. monary dysplasia with metaanalytic techniques. Pediatr. Infect. Dis. Obstet. 79: 209–215. J. 24: 1033–1039. Simecka, J.W., J.K. Davis, M.K. Davidson, S.R. Ross, C.T.K.-H. Städtlander Schilling, V. 1928. Eperythrozoon coccoides, eine neue durch Splenektomie and G.H. Cassell. 1992. Mycoplasma diseases of animals. In Mycoplas- aktivierbare Dauerinfektion der weissen Maus. Klin. Wochenschr. 7: mas: Molecular Biology and Pathogenesis (edited by Maniloff, McEl- 1854–1855. haney, Finch, and Baseman). American Society for Microbiology, Schmidhauser, C., R. Dudler, T. Schmidt and R.W. Parish. 1990. A myco- Washington, D.C., pp. 391–415. plasmal protein influences tumour cell invasiveness and contact inhi- Singh, K.C. and P.K. Uppal. 1987. Isolation of Mycoplasma gallinarum bition in vitro. J. Cell Sci. 95: 499–506. from sheep. Vet. Rec. 120: 464. Schmitt, K., W. Daubener, D. Bitter-Suermann and U. Hadding. 1988. Singh, Y., D.N. Garg, P.K. Kapoor and S.K. Mahajan. 2004. Isolation of A safe and efficient method for elimination of cell culture mycoplas- Mycoplasma bovoculi from genitally diseased bovines and its experi- mas using ciprofloxacin. J. Immunol. Methods 109: 17–25. mental pathogenicity in pregnant guinea pigs. Indian J. Exp. Biol. Schoeb, T.R. 2000. Respiratory diseases of rodents. Vet. Clin. North Am. 42: 933–936. Exot. Anim. Pract. 3: 481–496, vii. Slatter, D.H. 2001. Fundamentals of Veterinary Ophthamology, 3rd Scudamore, J.M. 1976. Observations on the epidemiology of contagious edn. Elsevier Health Sciences, Saint Louis, MO. bovine pleuropneumonia: Mycoplasma mycoides in urine. Res. Vet. Sci. Smith, A. and J. Mowles. 1996. Prevention and control of Mycoplasma 20: 330–333. infection of cell cultures. In Molecular and Diagnostic Procedures in Seto, S., G. Layh-Schmitt, T. Kenri and M. Miyata. 2001. Visualization of Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, the attachment organelle and cytadherence proteins of Mycoplasma San Diego, pp. 445–451. pneumoniae by immunofluorescence microscopy. J. Bacteriol. 183: Smith, D., W. Russell, W. Ingledew and D. Thirkell. 1993. Hydrolysis of 1621–1630. urea by Ureaplasma urealyticum generates a transmembrane potential Seybert, A., R. Herrmann and A.S. Frangakis. 2006. Structural analy- with resultant ATP synthesis. J. Bacteriol. 175: 3253–3258. sis of Mycoplasma pneumoniae by cryo-electron tomography. J. Struct. Smith, P. 1986. Mass cultivation of ureaplasmas and some applications. Biol. 156: 342–354. Pediatr. Infect. Dis. 5: S313–315. Shahram, M., R.A. Nicholas, A.P. Wood and D.P. Kelly. 2010. Further Smith, P.F. 1992. Membrane lipid and lipopolysaccharide structures. evidence to justify reassignment of Mycoplasma mycoides subspecies In Mycoplasmas: Molecular Biology and Pathogenesis (edited by mycoides Large Colony type to Mycoplasma mycoides subspecies capri. Maniloff, McElhaney, Finch and Baseman). American Society for Syst. Appl. Microbiol. 33: 20–24. Microbiology, Washington, D.C., pp. 79–91. Shepard, M. 1954. The recovery of pleuropneumonia-like organisms So, A.K., P.M. Furr, D. Taylor-Robinson and A.D. Webster. 1983. Arthri- from Negro men with and without nongonococcal urethritis. Am. J. tis caused by Mycoplasma salivarium in hypogammaglobulinaemia. Br. Syph. Gonorrhea Vener. Dis. 38: 113–124. Med. J. (Clin. Res. Ed.) 286: 762–763. Shepard, M. 1967. Cultivation and properties of T-strains of Mycoplasma Somerson, N.L., D. Taylor-Robinson and R.M. Chanock. 1963. associated with nongonococcal urethritis. Ann. N. Y. Acad. Sci. 143: Hemolyin production as an aid in the identification and quan- 505–514. titation of Eaton agent (Mycoplasma pneumoniae). Am. J. Hyg. 77: Shepard, M. and C. Lunceford. 1967. Occurrence of urease in T strains 122–128. of Mycoplasma. J. Bacteriol. 93: 1513–1520. Somerson, N.L. and B.C. Cole. 1979. The Mycoplasma flora of human Shepard, M. and D. Howard. 1970. Identification of “T” mycoplasmas and non-human primates. In The Mycoplasmas, vol. 1 (edited by in primary agar cultures by means of a direct test for urease. Ann. N. Tully and Whitcomb). Academic Press, New York, pp. 191–216. Y. Acad. Sci. 174: 809–819. Somerson, N.L., J.P. Kocka, D. Rose and R.A. Del Giudice. 1982. Shepard, M.C., C.D. Lunceford, D.K. Ford, R.H. Purcell, D. Taylor- ­Isolation of acholeplasmas and a Mycoplasma from vegetables. Appl. ­Robinson, S. Razin and F.T. Black. 1974. Ureaplasma urealyticum gen. Environ. Microbiol. 43: 412–417. nov., sp. nov.: proposed nomenclature for Human-T (T-strain) myco- Spergser, J., C. Aurich, J.E. Aurich and R. Rosengarten. 2002. High plasmas. Int. J. Syst. Bacteriol. 24: 160–171. prevalence of mycoplasmas in the genital tract of asymptomatic stal- Shepard, M.C. and G.K. Masover. 1979. Special features of ureaplasmas. lions in Austria. Vet. Microbiol. 87: 119–129. In The Mycoplamas, vol. 1 (edited by Barile and Razin). Academic Spergser, J., S. Langer, S. Muck, K. Macher, M. Szostak, R. Rosengarten Press, New York, pp. 452–494. and H.-J. Busse. 2010. Mycoplasma mucosicanis sp. nov., isolated from Genus II. Ureaplasma 637

the mucosa of dogs. Int. J. Syst. Evol. Microbiol.: published 23 April treatment, on Mycoplasma haemofelis infection. Vet. Microbiol. 2010 as doi:10.1099/ijs.0.015750-0. 117: 169–179. Splitter, E.J. 1950. Eperythrozoon suis, the etiologic agent of icteroane- Taylor-Robinson, D., J. Canchola, H. Fox and R.M. Chanock. 1964. A mia–an anaplasmosis-like disease in swine. Am. J. Vet. Res. 11: 324– newly identified oral Mycoplasma (M. orale) and its relationship to 329. other human mycoplasmas. Am. J. Hyg. 80: 135–148. Spooner, R., W. Russell and D. Thirkell. 1992. Characterization of the Taylor-Robinson, D. and Z. Dinter. 1968. Unexpected serotypes of immunoglobulin A protease of Ureaplasma urealyticum. Infect. Immun. mycoplasmas isolated from pigs. J. Gen. Microbiol. 53: 221–229. 60: 2544–2546. Taylor-Robinson, D., G.W. Csonka and M.J. Prentice. 1977. Ståby, M. 2004. Seal finger-a problem among hunters once again. Human intra-urethral inoculation of ureplasmas. Q. J. Med. 46: Lakartidningen 101: 1910–1911. 309–326. Stemke, G. and J. Robertson. 1981. Modified colony indirect epifluo- Taylor-Robinson, D. and W.M. McCormack. 1979. Mycoplasmas in rescence test for serotyping Ureaplasma urealyticum and an adapta- human genitourinary infections. In The Mycoplasmas, Volume 2, tion to detect common antigenic specificity. J. Clin. Microbiol. 14: Human and Animal Mycoplasmas (edited by Tully and Whitcomb). 582–584. Academic Press, New York, pp. 308–357. Stemke, G. and J. Robertson. 1982. Comparison of two methods for Taylor-Robinson, D. 1983a. Recovery of mycoplasmas from the genito- enumeration of mycoplasmas. J. Clin. Microbiol. 16: 959–961. urinary tract. In Methods in Mycoplasmology, vol. 2 (edited by Razin Stemke, G.W. and J.A. Robertson. 1985. Problems associated with sero- and Tully). Academic Press, New York, pp. 19–26. typing strains of Ureaplasma urealyticum. Diagn. Microbiol. Infect. Dis. Taylor-Robinson, D. 1983b. Metabolism inhibition tests. In Methods in 3: 311–320. Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, Stemke, G., M. Stemler and J. Robertson. 1984. Growth characteristics New York, pp. 411–421. of ureaplasmas from animal and human sources. Isr. J. Med. Sci. 20: Taylor-Robinson, D. and R.N. Gourlay. 1984. Genus II. Ureaplasma 935–937. Shepard, Lunceford, Ford, Purcell, Taylor-Robinson, Razin and Stemler, M.E., G.W. Stemke and J.A. Robertson. 1987. ATP mea- Black 1974. In Bergey’s Manual of Systematic Bacteriology, 8th edn, surements obtained by luminometry provide rapid estimation of vol. 1 (edited by Kreig and Holt). Williams & Wilkins, Baltimore, pp. Ureaplasma­ urealyticum growth. J. Clin. Microbiol. 25: 427–429. 770–775. Stenske, K.A., D.A. Bemis, K. Hill and D.J. Krahwinkel. 2005. Acute Taylor-Robinson, D. 1985. Mycoplasmal and mixed infections of the polyarthritis and septicemia from Mycoplasma edwardii after surgical human male urogenital tract and their possible complications. In removal of bilateral adrenal tumors in a dog. J. Vet. Intern. Med. 19: The Mycoplasmas, vol. 4 (edited by Razin and Barile). Academic 768–771. Press, New York, pp. 27–63. Stipkovits, L., Z. Varga, M. Dobos-Kovacs and M. Santha. 1984a. Bio- Taylor-Robinson, D. and P. Furr. 1986. Clinical antibiotic resistance of chemical and serological examination of some Mycoplasma strains of Ureaplasma urealyticum. Pediatr. Infect. Dis. 5: S335–337. goose origin. Acta Vet. Hung. 32: 117–125. Taylor-Robinson, D. and P.M. Furr. 1997. Genital Mycoplasma infections. Stipkovits, L., Z. Varga, G. Laber and J. Bockmann. 1984b. A compari- Wien. Klin. Wochenschr. 109: 578–583. son of the effect of tiamulin hydrogen fumarate and tylosin tartrate Taylor-Robinson, D. and P. Horner. 2001. The role of Mycoplasma geni- on mycoplasmas of ruminants and some animal ureaplasmas. Vet. talium in non-gonococcal urethritis. Sex. Transm. Infect. 77: 229– Microbiol. 9: 147–153. 231. Stoffregen, W.C., D.P. Alt, M.V. Palmer, S.C. Olsen, W.R. Waters and J.A. Taylor-Robinson, D., C. Gilroy, B. Thomas and P. Hay. 2004. Mycoplasma Stasko. 2006. Identification of a Haemomycoplasma species in anemic genitalium in chronic non-gonococcal urethritis. Int. J. STD AIDS 15: reindeer (Rangifer tarandus). J. Wildl. Dis. 42: 249–258. 21–25. Swensen, C., J. VanHamont and B.S. Dunbar. 1983. Specific protein dif- Taylor, R.R., H. Varsani and R.J. Miles. 1994. Alternatives to arginine ferences among strains of Ureaplasma urealyticum as determined by as energy sources for the non-fermentative Mycoplasma gallinarum. two-dimensional gel electrophoresis and a sensitive silver stain. Int. J. FEMS Microbiol. Lett. 115: 163–167. Syst. Bacteriol. 33: 417–421. Teng, L., X. Zheng, J. Glass, H. Watson, J. Tsai and G. Cassell. 1994. Switzer, W.P. 1955. Studies on infectious atrophic rhinitis. IV. Character- Ureaplasma urealyticum biovar specificity and diversity are encoded ization of a pleuropneumonia-like organism isolated from the nasal in multiple-banded antigen gene. J. Clin. Microbiol. 32: 1464– cavities of swine. Am. J. Vet. Res. 16: 540–554. 1469. Sykes, J.E., L.M. Ball, N.L. Bailiff and M.M. Fry. 2005. ‘Candidatus Myco- ter Laak, E.A., J.H. Noordergraaf and M.H. Verschure. 1993. Sus- plasma haematoparvum’, a novel small haemotropic mycoplasma ceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma from a dog. Int. J. Syst. Evol. Microbiol. 55: 27–30. diversum strains to antimicrobial agents in vitro. Antimicrob. Agents Sykes, J.E., N.L. Drazenovich, L.M. Ball and C.M. Leutenegger. 2007. Chemother. 37: 317–321. Use of conventional and real-time polymerase chain reaction to ter Laak EA, Noordergraaf JH and Boomsluiter E. 1992a. The nasal determine the epidemiology of hemoplasma infections in anemic mycoplasmal flora of healthy calves and cows. Zentralbl Veteri- and nonanemic cats. J. Vet. Intern. Med. 21: 685–693. narmed B 39: 610–616. Tagawa, M., K. Matsumoto and H. Inokuma. 2008. Molecular detection ter Laak EA, Noordergraaf JH and Dieltjes RP. 1992b. Prevalence of of Mycoplasma wenyonii and ‘Candidatus Mycoplasma haemobos’ in mycoplasmas in the respiratory tracts of pneumonic calves. Zentralbl cattle in Hokkaido, Japan. Vet. Microbiol. 132: 177–180. Veterinarmed B 39: 553–562. Tasker, S., S.H. Binns, M.J. Day, T.J. Gruffydd-Jones, D.A. Harbour, C.R. Thiaucourt, F., G. Bolske, B. Leneguersh, D. Smith and H. Wesonga. Helps, W.A. Jensen, C.S. Olver and M.R. Lappin. 2003. Use of a PCR 1996. Diagnosis and control of contagious caprine pleuropneumo- assay to assess the prevalence and risk factors for Mycoplasma haemofe- nia. Rev. Sci. Tech. 15: 1415–1429. lis and ‘Candidatus Mycoplasma haemominutum’ in cats in the United Thirkell, D., A. Myles and W. Russell. 1989. Serotype 8- and serocluster- Kingdom. Vet. Rec. 152: 193–198. specific surface-expressed antigens of Ureaplasma urealyticum. Infect. Tasker, S., S.M. Caney, M.J. Day, R.S. Dean, C.R. Helps, T.G. Immun. 57: 1697–1701. Knowles, P.J. Lait, M.D. Pinches and T.J. Gruffydd-Jones. 2006. Effect of chronic FIV infection, and efficacy of marbofloxacin 638 Family I. Mycoplasmataceae

Thirkell, D., A. Myles and D. Taylor-Robinson. 1990. A comparison of cedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Aca- four major antigens in five human and several animal strains of urea­ demic Press, San Diego, pp. 33–39. plasmas. J. Med. Microbiol. 32: 163–168. Tully, J.G. and S. Razin (editors). 1996. Molecular and Diagnostic Thomsen, A.C. 1974. The isolation of Mycoplasma primatum during an Procedures in Mycoplasmology, vol. 2. Academic Press, San Diego, autopsy study of the Mycoplasma flora of the human urinary tract. Acta CA. Pathol. Microbiol. Scand. B Microbiol. Immunol. 82B: 653–656. Tyzzer, E.E. and D. Weinman. 1939. Haemobartonella n.g. (Bartonella olim Thurston, J.P. 1953. The chemotherapy of Eperythrozoon coccoides (Schil- pro parte) H. microti n. sp. of the field vole, Microtus pennsylvanicus. ling 1928). Parasitology 43: 170–174. Am. J. Hyg. 30: 141–157. Tiong, S.K. 1990. Mycoplasmas and acholeplasmas isolated from Uilenberg, G., F. Thiaucourt and F. Jongejan. 2004. On molecular tax- ducks and their possible association with pasteurellas. Vet. Rec. 127: onomy: what is in a name? Exp. Appl. Acarol. 32: 301–312. 64–66. Uilenberg, G., F. Thiaucourt and F. Jongejan. 2006. Mycoplasma and Totten, P.A., M.A. Schwartz, K.E. Sjostrom, G.E. Kenny, H.H. Hands- Eperythrozoon (Mycoplasmataceae). Comments on a recent paper. Int. field, J.B. Weiss and W.L. Whittington. 2001. Association of Myco- J. Syst. Evol. Microbiol. 56: 13–14. plasma genitalium with nongonococcal urethritis in heterosexual Vancini, R.G. and M. Benchimol. 2008. Entry and intracellular location men. J. Infect. Dis. 183: 269–276. of Mycoplasma hominis in Trichomonas vaginalis. Arch. Microbiol. 189: Trüper, H.G. and L. de’Clari. 1998. Taxonomic note: erratum and cor- 7–18. rection of further specific epithets formed as substantives (nouns) Vasconcellos Cardosa, M., A. Blanchard, S. Ferris, R. Verlengia, J. “in apposition”. Int. J. Syst. Bacteriol. 48: 615. Timenetsky and R.A. Florio Da Cunha. 2000. Detection of Ureaplasma Tryon, V.V. and J.B. Baseman. 1992. Pathogenic determinants and diversum in cattle using a newly developed PCR-based detection assay. mechanisms. In Mycoplasmas: Molecular Biology and Pathogenesis Veterinary Microbiology 72: 241–250. (edited by Maniloff, McElhaney, Finch and Baseman). American Vilei, E.M., J. Nicolet and J. Frey. 1999. IS1634, a novel insertion ele- Society for Microbiology, Washington, D.C., pp. 457–471. ment creating long, variable-length direct repeats which is specific Tu, A.H., L.L. Voelker, X. Shen and K. Dybvig. 2001. Complete nucle- for Mycoplasma mycoides subsp. mycoides small-colony type. J. Bacteriol. otide sequence of the Mycoplasma virus P1 genome. Plasmid 45: 181: 1319–1323. 122–126. Voelker, L.L., K.E. Weaver, L.J. Ehle and L.R. Washburn. 1995. Associa- Tully, J.G. and I. Ruchman. 1964. Recovery, identification, and neu- tion of lysogenic bacteriophage MAV1 with virulence of Mycoplasma rotoxicity of Sabin’s Type A and C mouse Mycoplasma (PPLO) arthritidis. Infect. Immun. 63: 4016–4023. from lyophilized cultures. Proc. Soc. Exp. Biol. Med. 115: 554– Voelker, L.L. and K. Dybvig. 1999. Sequence analysis of the Mycoplasma 558. arthritidis bacteriophage MAV1 genome identifies the putative viru- Tully, J.G., M.F. Barile, R.A. Del Giudice, T.R. Carski, D. Armstrong lence factor. Gene 233: 101–107. and S. Razin. 1970. Proposal for classifying strain PG-24 and related Vogelzang, A. and G. Compeer-Dekker. 1969. Elimination of Myco- canine mycoplasmas as Mycoplasma edwardii sp. n. J. Bacteriol. 101: plasma from various cell cultures. Antonie Van Leeuwenhoek 35: 346–349. 393–408. Tully, J.G., M.F. Barile, D.G.F. Edward, T.S. Theodore and H. Erno. Waites, K., D. Crouse and G. Cassell. 1992. Antibiotic susceptibilities 1974. Characterization of some caprine mycoplasmas, with proposals and therapeutic options for Ureaplasma urealyticum infections in neo- for new species, Mycoplasma capricolum and Mycoplasma putrefaciens. J. nates. Pediatr. Infect. Dis. J. 11: 23–29. Gen. Microbiol. 85: 102–120. Waites, K., C.M. Bébéar, J.A. Robertson, D.F. Talkington and G.E. Kenny. Tully, J.G. and R.F. Whitcomb. 1979. The Mycoplasmas Volume II: 2001. Laboratory Diagnosis of Mycoplasmal Infections. Cumitech 34. Human and Animal Mycoplasmas. Academic Press, New York. ASM Press, Washington, D.C. Tully, J.G. 1983. Cloning and filtration techniques for mycoplasmas. In Waites, K. and D. Talkington. 2005. New developments in human Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Aca- disease due to mycoplasmas. In Mycoplasmas: Molecular Biol- demic Press, New York, pp. 173–177. ogy, Pathogenicity, and Strategies for Control (edited by Tully, J.G. and S.E. Razin. 1983. Methods in Mycoplasmology, vol. 2. Blanchard and Browning). Horizon Bioscience, Norfolk, UK, Academic Press, New York. pp. 289–354. Tully, J.G., D. Taylor-Robinson, D.L. Rose, R.M. Cole and J.M. Bové. Waites, K.B., P.T. Rudd, D.T. Crouse, K.C. Canupp, K.G. Nelson, C. 1983. Mycoplasma genitalium, a new species from the human urogeni- Ramsey and G.H. Cassell. 1988. Chronic Ureaplasma urealyticum and tal tract. Int. J. Syst. Bacteriol. 33: 387–396. Mycoplasma hominis infections of central nervous system in preterm Tully, J.G. 1993. Current status of the mollicute flora of humans. Clin. infants. Lancet 1: 17–21. Infect. Dis. 17 Suppl 1: S2–S9. Waites, K.B., T.A. Figarola, T. Schmid, D.M. Crabb, L.B. Duffy and J.W. Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised tax- Simecka. 1991. Comparison of agar versus broth dilution techniques onomy of the class Mollicutes - proposed elevation of a monophyletic for determining antibiotic susceptibilities of Ureaplasma urealyticum. cluster of arthropod-associated Mollicutes to ordinal rank (Entomoplas- Diagn. Microbiol. Infect. Dis. 14: 265–271. matales ord. nov.), with provision for familial rank to separate species Washburn, L.R., L.L. Voelker, L.J. Ehle, S. Hirsch, C. Dutenhofer, with nonhelical morphology (Entomoplasmataceae fam. nov.) from K. Olson and B. Beck. 1995. Comparison of Mycoplasma arthri- helical species (Spiroplasmataceae), and emended descriptions of the tidis strains by enzyme-linked immunosorbent assay, immuno- order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. blotting, and DNA restriction analysis. J. Clin. Microbiol. 33: 43: 378–385. 2271–2279. Tully, J.G. 1995a. International Committee on Systematic Bacteriology Watson, H.L., D.K. Blalock and G.H. Cassell. 1990. Variable antigens Subcommittee on the Taxonomy of Mollicutes. Revised minimal stan- of Ureaplasma urealyticum containing both serovar-specific and dards for description of new species of the class Mollicutes. Int. J. Syst. serovar-cross-reactive epitopes. Infect. Immun. 58: 3679–3688. Bacteriol. 45: 605–612. Webster, A.D., D. Taylor-Robinson, P.M. Furr and G.L. Asherson. 1978. Tully, J.G. 1995b. Culture medium formulation for primary isolation Mycoplasmal (Ureaplasma) septic arthritis in hypogammaglobulinae- and maintenance of mollicutes. In Molecular and Diagnostic Pro- mia. Br. Med. J. 1: 478–479. Family II. Incertae sedis 639

Webster, D., H. Windsor, C. Ling, D. Windsor and D. Pitcher. 2003. Chronic Woodson, B.A., K.S. McCarty and M.C. Shepard. 1965. Arginine metab- bronchitis in immunocompromised patients: association with a novel olism in Mycoplasma and infected L929 fibroblasts. Arch. Biochem. Mycoplasma species. Eur. J. Clin. Microbiol. Infect. Dis. 22: 530–534. 109: 364–371. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Woubit, S., S. Lorenzon, A. Peyraud, L. Manso-Silván and F. Thiau- Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylo- court. 2004. A specific PCR for the identification of Mycoplasma genetic analysis of the mycoplasmas: basis for their classification. J. capricolum subsp. capripneumoniae, the causative agent of conta- Bacteriol. 171: 6455–6467. gious caprine pleuropneumonia (CCPP). Vet. Microbiol. 104: Welchman, D.B., J. Bradbury, D. Cavanagh and N. Aebischer. 2002. 125–132. Infectious agents associated with respiratory disease in pheasants. Wroblewski, W. 1931. Morphologie et cycle évolutif des microbnes de la Vet. Rec. 150: 658–664. péripneumonie das bovides et de l’agalaxie contagieuse des chêvres Wellehan, J.F., M. Calsamiglia, D.H. Ley, M.S. Zens, A. Amonsin and V. et des moutons. Ann. Inst. Pasteur (Paris) 47: 94–115. Kapur. 2001. Mycoplasmosis in captive crows and robins from Min- Xie, X. and J. Zhang. 2006. Trends in the rates of resistance of Ure- nesota. J. Wildl. Dis. 37: 547–555. aplasma urealyticum to antibiotics and identification of the mutation Westberg, J., A. Persson, B. Pettersson, M. Uhlen and K.E. Johansson. site in the quinolone resistance-determining region in Chinese 2002. ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp. patients. FEMS Microbiol. Lett. 259: 181–186. mycoides small colony type. FEMS Microbiol. Lett. 208: 207–213. Yagihashi, T., T. Nunoya and Y. Otaki. 1983. Effects of dual infection Whitescarver, J. and G. Furness. 1975. T-mycoplasmas: a study of the of chickens with Mycoplasma synoviae and Mycoplasma gallinaceum or morphology, ultrastructure and mode of division of some human infectious bursal disease virus on infectious synovitis. Nippon Juigaku strains. J. Med. Microbiol. 8: 349–355. Zasshi 45: 529–532. Whittlestone, P. 1979. Porcine mycoplasmas. In The Mycoplasmas, Yamamoto, R., C.H. Bigland and H.B. Ortmayer. 1965. Characteristics vol. 2 (edited by Tully and Whitcomb). Academic Press, New York, of Mycoplasma meleagridis sp. n. isolated from turkeys. J. Bacteriol. 90: pp. 133–176. 47–49. Wieslander, A., M.J. Boyer and H. Wróblewski. 1992. Membrane pro- Yanez, A., L. Cedillo, O. Neyrolles, E. Alonso, M.C. Prevost, J. Rojas, tein structure. In Mycoplasmas: Molecular Biology and Pathogenesis H.L. Watson, A. Blanchard and G.H. Cassell. 1999. Mycoplasma pen- (edited by Maniloff, McElhaney, Finch and Baseman). American etrans bacteremia and primary antiphospholipid syndrome. Emerg. Society for Microbiology, Washington, D.C., pp. 93–112. Infect. Dis. 5: 164–167. Willi, B., F.S. Boretti, C. Baumgartner, S. Tasker, B. Wenger, V. Cattori, Yechouron, A., J. Lefebvre, H.G. Robson, D.L. Rose and J.G. Tully. 1992. M.L. Meli, C.E. Reusch, H. Lutz and R. Hofmann-Lehmann. 2006a. Fatal septicemia due to Mycoplasma arginini: a new human zoonosis. Prevalence, risk factor analysis, and follow-up of infections caused by Clin. Infect. Dis. 15: 434–438. three feline hemoplasma species in cats in Switzerland. J. Clin. Micro- Yoder, H.W. and M.S. Hofstad. 1964. Characterization of avian myco- biol. 44: 961–969. plasmas. Avian Dis. 8: 481–512. Willi, B., S. Tasker, F. S. Boretti, M. G. Doherr, V. Cattori, M. L. Meli, R. Yogev, D., S. Levisohn and S. Razin. 1989. Genetic and antigenic relat- G. Lobetti, R. Malik, C. E. Reusch, H. Lutz, R. Hofmann-Lehmann. edness between Mycoplasma gallisepticum and Mycoplasma synoviae. 2006b. Phylogenetic analysis of “Candidatus Mycoplasma turicensis” Vet. Microbiol. 19: 75–84. isolates from pet cats in the United Kingdom, Australia, and South Zheng, X., L. Teng, H. Watson, J. Glass, A. Blanchard and G. Cassell. Africa, with analysis of risk factors for infection. J. Clin. Microbiol. 1995. Small repeating units within the Ureaplasma urealyticum MB 44: 4430–4435. antigen gene encode serovar specificity and are associated with anti- Windsor, R.S. and W.N. Masiga. 1977. Investigations into the role of car- gen size variation. Infect. Immun. 63: 891–898. rier animals in the spread of contagious bovine pleuropneumonia. Zheng, X., D.A. Olson, J.G. Tully, H.L. Watson, G.H. Cassell, D.R. Res. Vet. Sci. 23: 224–229. Gustafson, K.A. Svien and T.F. Smith. 1997. Isolation of Myco­ Woods, J.E., M.M. Brewer, J.R. Hawley, N. Wisnewski and M.R. Lappin. plasma hominis from a brain abscess. J. Clin. Microbiol. 35: 2005. Evaluation of experimental transmission of Candidatus Myco- 992–994. plasma haemominutum and Mycoplasma haemofelis by Ctenocephalides felis to cats. Am. J. Vet. Res. 66: 1008–1012.

Family II. Incertae sedis

Da n i e l R. Br o w n , Sé v e r i n e Ta s k e r , Jo a n n e B. Me s s i c k a n d Ha r o l d Ne i m a rk This family accommodates the genera Eperythrozoon and Haemo- any species. These organisms are now known to be unambigu- bartonella. These wall-less hemotropic bacteria were once placed ously affiliated with the order Mycoplasmatales on the basis of 16S in the family Anaplasmataceae, order Rickettsiales, because they are rRNA similarities, plus morphology, DNA G+C contents, and evi- obligate blood parasites. None have been cultivated on artificial dence that they use the codon UGA to encode tryptophan (Ber- media, so no type strains have been established. Motility and bio- ent and Messick, 2003), but their nomenclature remains a matter chemical parameters have not been definitively established for of controversy (Neimark et al., 2005; Uilenberg et al., 2006). 640 Family II. Incertae sedis

Genus I. Eperythrozoon Schilling 1928, 293AL

Da n i e l R. Br o w n , Sé v e r i n e Ta s k e r , Jo a n n e B. Me ss i c k a n d Ha r o l d Ne i m a r k E.pe.ry.thro.zo¢on. Gr. pref. epi on; Gr. adj. erythros red; Gr. neut. n. zoon living being, animal; N.L. neut. n. Eperythrozoon (presumably intended to mean) animals on red (blood cells). Cells adherent to host erythrocyte surfaces are coccoid and about morphology and regular association with vertebrate hosts 350 nm in diameter, but may arrange to appear as chains or ­support exclusion from the genera Spiroplasma, Entomoplasma, deform to appear rod- or ring-shaped in stained blood smears. and Mesoplasma, but sterol requirement, the degree of aerobio- Type species: Eperythrozoon coccoides Schilling 1928, 1854. sis, and the capacity to hydrolyze arginine, characteristics that would help to confirm their provisional 16S rRNA-based place- Further descriptive information ment in the genus Mycoplasma, remain unknown. Hemotropic mollicutes such as the species formerly called Eperythrozoon coccoides (trivial name, hemoplasmas; Neimark Taxonomic comments et al., 2005) infect a variety of mammals occasionally includ- The taxonomy and nomenclature of the uncultivated hemotro- ing humans. Transmission can be through ingestion of infected pic bacteria originally assigned to the genus Eperythrozoon remain blood, percutaneous inoculation, or by arthropod vectors matters of current controversy. It is now undisputed that, on (Sykes et al., 2007; Willi et al., 2006). The pathogenicity of dif- the basis of their lack of a cell wall, small cell size, low G+C ferent hemoplasma species is variable, and strain virulence and content, use of the codon UGA to encode tryptophan, regular host immunocompetence likely play roles in the development association with vertebrate hosts, and 16S rRNA gene sequences of disease. Clinical syndromes range from acute fatal hemolytic that are most similar (80–84%) to species in the pneumoniae anemia to chronic insidious anemia. Signs may include ane- group of genus Mycoplasma, these organisms are properly affili- mia, pyrexia, anorexia, dehydration, weight loss, and infertil- ated with the Mycoplasmatales. However, the proposed trans- ity. The presence of erythrocyte-bound antibodies has been fers of Eperythrozoon and Haemobartonella species to the genus demonstrated in some hemoplasma-infected animals and may Mycoplasma (Neimark et al., 2001, 2005) were opposed on the contribute to anemia. Animals can remain chronic asymptom- grounds that the degree of 16S rRNA gene sequence similarity atic carriers of hemoplasmas after acute infection. PCR is the is insufficient (Uilenberg et al., 2004, 2006). The alternative of diagnostic test of choice for hemoplasmosis. Tetracycline treat- situating the hemoplasmas in a new genus in the Mycoplasmata- ment reduces the number of organisms in peripheral blood, ceae (Uilenberg et al., 2006) would regrettably compound the but probably does not eradicate the organisms from infected 16S rRNA gene-based polyphyly within Mycoplasma on no other animals. basis than a capacity to adhere to the surface of erythrocytes in vivo. Enrichment and isolation procedures The proposed transfer of the type species Eperythrozoon Hemoplasmas have not yet been successfully grown in continu- ­coccoides to the genus Mycoplasma (Neimark et al., 2005) is ous culture in vitro, although recent work (Li et al., 2008) sug- ­complicated by priority because Eperythrozoon predates Myco- gests that in vitro maintenance of the species Mycoplasma suis plasma. However, the alternative of uniting the genera by trans- may be possible. ferring all mycoplasmas to the genus Eperythrozoon is completely ­unjustifiable considering the biological characteristics of the Maintenance procedures non-hemotropic majority of Mycoplasma species. The Judicial Hemoplasmas can be frozen in heparin- or EDTA-anticoagu- Commission of the International Committee on Systematics of lated blood cryopreserved with dimethylsulfoxide. Prokaryotes declined to rule on a request for an opinion in this matter during their 2008 meeting, but a provisional placement Differentiation of the genus Eperythrozoon of the former Eperythrozoon species in the genus Mycoplasma has from other genera otherwise been embraced by specialists in the molecular biology A distinctive characteristic of these organisms is that they are and clinical pathogenicity of these and similar hemotropic found only in the blood of vertebrate hosts or transiently in organisms. At present, the designation “Candidatus Mycoplasma” arthropod vectors of transmission. The tenuous distinction must still be used for new types. between species of Eperythrozoon and those of Haemobartonella Further reading was based on the relatively more common visualization of eperythrozoa as ring forms (now known to be artifactual) in Kreier, J.P. and M. Ristic. 1974. Genus IV. Haemobartonella Tyzzer AL stained blood smears and the perception that eperythrozoa were and Weinman 1939, 143 ; Genus V. Eperythrozoon Schilling AL observed with about equal frequency on erythrocytes and free in 1928, 1854 . In Bergey’s Manual of Determinative Bacteriol- plasma, while haemobartonellae were thought to occur less ogy, 8th edn (edited by Buchanan and Gibbons). Williams & often free in plasma. Properties that partially fulfill criteria for Wilkins, Baltimore, pp. 910–914. assignment of this genus to the class Mollicutes (Brown et al., Differentiation of the species of the genus 2007) include absence of a cell wall, filterability, and the ­presence Eperythrozoon of conserved 16S rRNA gene sequences. Presumptive use of the codon UGA to encode tryptophan (Berent and ­Messick, 2003) Species differentiation relies principally on 16S rRNA gene supports exclusion from the genera Anaeroplasma­ , Asteroleplasma, sequencing. Some species exhibit a degree of host specificity, Acholeplasma, and “Candidatus Phytoplasma”. ­Non-­spiral cellular although cross-infection of related hosts has been reported. Genus I. Eperythrozoon 641

List of species of the genus Eperythrozoon 1. Mycoplasma coccoides (Schilling 1928) Neimark, Peters, Source: observed in association with erythrocytes or unat- Robinson and Stewart 2005, 1389VP (Eperythrozoon coccoides tached in suspension in the blood of sheep, goats, and Schilling 1928, 1854) rarely in eland and splenectomized deer. coc.co′ides. N.L. masc. n. coccus (from Gr. masc. n. kokkos DNA G+C content (mol%): not determined. grain, seed) coccus; L. suff. -oides (from Gr. suff. eides from Type strain: not established. Gr. n. eidos that which is seen, form, shape, figure), resem- Sequence accession no. (16S rRNA gene): AF338268. bling, similar; N.L. neut. adj. coccoides coccus-shaped. 4. Mycoplasma suis corrig. (Splitter 1950) Neimark, ­Johansson, Pathogenic; causes anemia in wild and captive mice, and Rikihisa and Tully 2002, 683VP (Eperythrozoon suis Splitter captive rats, hamsters, and rabbits. Transmission is believed to 1950, 513) be vector-borne and mediated by the rat louse Polyplex spinu- su¢is. L. gen. n. suis of the pig. losa and the mouse louse Polyplex serrata. Neoarsphenamine and oxophenarsine were thought to be effective chemother- Cells are coccoid. Motility for this species has not been apeutic agents for treatment of Mycoplasma coccoides infection assessed. This species has not been grown on any artificial in captive rodents, whereas tetracyclines are effective only at medium; therefore, notable biochemical parameters are keeping infection at subclinical levels (Thurston, 1953). not known. Source: observed in association with the erythrocytes of Neoarsphenamine and tetracyclines are effective thera- wild and captive rodents. peutic agents. An enzyme-linked immunosorbant assay DNA G+C content (mol%): not determined. (ELISA) and PCR-based detection assays to enable diagnosis Type strain: not established. of infection have been described (Groebel et al., 2009; Gwalt- Sequence accession no. (16S rRNA gene): AY171918. ney and Oberst, 1994; Hoelzle, 2008; Hsu et al., 1992). Pathogenic; causes febrile icteroanemia in pigs. Trans- AL 2. Eperythrozoon parvum Splitter 1950, 513 mission occurs via insect vectors including Stomoxys calci- par¢vum. L. neut. adj. parvum small. trans and Aedes aegypti (Prullage et al., 1993). A nonpathogenic epierythrocytic parasite of pigs. Organic Source: observed in association with the erythrocytes of pigs. arsenicals are effective; tetracyclines suppress infection. DNA G+C content (mol%): not determined. Transmissible by parenteral inoculation and sometimes by Type strain: not established. massive oral inoculation. This is the only remaining species Sequence accession no. (16S rRNA gene): AF029394. of Eperythrozoon whose name has standing in nomenclature Further comment: the original spelling of the specific that has not yet been examined by molecular genetic meth- ­epithet, haemosuis (sic), has been corrected by the List ods. It seems likely that, if a specimen of this organism can ­Editor. be found, it will prove to be a mycoplasma. 5. Mycoplasma wenyonii (Adler and Ellenbogen 1934) Nei- VP 3. Mycoplasma ovis (Neitz, Alexander and de Toit 1934) Nei- mark, Johansson, Rikihisa and Tully 2002, 683 (Eperythro- mark, Hoff and Ganter 2004, 369VP (Eperythrozoon ovis Neitz, zoon wenyonii Adler and Ellenbogen 1934, 220) Alexander and de Toit 1934, 267) we.ny.o¢ni.i. N.L. masc. gen. n. wenyonii of Wenyon, named o¢vis. L. fem. gen. n. ovis of a sheep. after Charles Morley Wenyon (1878–1948), an investigator of these organisms. Cells are coccoid and motility for this species has not Cells are coccoid. Motility for this species has not been been assessed. The morphology of infected erythrocytes assessed. This species has not been grown on any artificial is altered demonstrating a marked depression at the site medium; therefore, notable biochemical parameters are of Myplasma ovis attachment. This species has not been not known. grown on artificial medium; therefore, notable biochemi- Pathogenic; causes anemia and subsequent lameness cal parameters are not known. and/or infertility in cattle. Transmission is primarily vector- Neoarsphenamine is an effective therapeutic agent. Myco- mediated by Dermacentor andersoni and reportedly can also plasma ovis is reported to share antigens with Mycoplasma occur vertically during gestation. Oxytetracycline is an effec- wenyonii (Kreier and Ristic, 1963), potentially complicating tive therapeutic agent (Montes et al., 1994). Mycoplasma serology-based diagnosis of infection. wenyonii is reported to share antigens with Mycoplasma ovis Pathogenic; causes mild to severe anemia in sheep and (Kreier and Ristic, 1963), potentially complicating serology- goats that often results in poor feed conversion. Transmis- based diagnosis of infection. sion occurs via blood-feeding arthropods, e.g., Haemophysa- Source: observed in association with the erythrocytes and lis plumbeum, Rhipicephalus bursa, Aedes camptorhynchus, and platelets of cattle (Kreier and Ristic, 1968). Culex annulirostris (Daddow, 1980; Howard, 1975; Nikol’skii DNA G+C content (mol%): not determined. and Slipchenko, 1969), and likely via fomites such as reused Type strain: not established. needles, shearing tools, and ear-tagging equipment (Brun- Sequence accession no. (16S rRNA gene): AF016546. Hansen et al., 1997; Mason and Statham, 1991). Species of unknown phylogenetic affiliation The phylogenetic affiliations of the following proposed organ- nomenclature. They are listed here merely because they have isms are unknown and their names do not have standing in been incidentally cited as species of Eperythrozoon. 642 Family II. Incertae sedis

1. “Eperythrozoon mariboi” Ewers 1971 attach to erythrocytes (Hoyte, 1962). The bodies ­differ from The name given to uncultivated polymorphic structures Mycoplasma wenyonii in morphology and include “frying-pan” observed on or in erythrocytes from flying foxes (Pteropus shaped structures. macrotis) following splenectomy (Ewers, 1971). The structures, 3. “Eperythrozoon tuomii” Tuomi and Von Bonsdorff 1967 described as fine lines, lines with rings, and rows of rings that span the diameter of the erythrocytes, differ from those of Uncultivated transmissible cell wall-less bodies observed in hemotropic mycoplasmas. Giemsa-stained blood smears and electron micrographs of blood from splenectomized calves. The bodies appeared in 2. “Eperythrozoon teganodes” Hoyte 1962 blood smears predominantly as delicate rings that did not The name given to uncultivated serially transmissible bod- attach to erythrocytes but were associated exclusively with ies observed in Giemsa-stained blood smears from ­cattle. thrombocytes (Tuomi and Von Bonsdorff, 1967; Uilenberg, The bodies only occur free in the blood plasma and do not 1967; Zwart et al., 1969).

Genus II. Haemobartonella Tyzzer and Weinman 1939, 305AL

Da n i e l R. Br o w n , Sé v e r i n e Ta s k e r , Jo a n n e B. Me ss i c k a n d Ha r o l d Ne i m a r k Ha.e.mo.bar.to.nel′la. Gr. n. haima (L. transliteration haema) blood; N.L. fem. n. Bartonella a bacterial genus; N.L. fem. n. Haemobartonella the blood (-inhabiting) Bartonella. Cells adherent to host erythrocyte surfaces are coccoid and Further descriptive information about 350 nm in diameter, but may occur as chains or deform Those organisms originally assigned to the genus Haemobartonella to appear rod- or ring-shaped in stained blood smears. are properly affiliated with the Mycoplasmatales, but their transfer Type species: Haemobartonella muris (Mayer 1921) Tyzzer and to the order has not yet been formalized. Any distinction between Weinman 1939AL (Bartonella muris Mayer 1921, 151; Bartonella Haemobartonella and Eperythrozoon is tenuous and possibly arbitrary muris ratti Regendanz and Kikuth 1928, 1578; Haemobartonella (Kreier and Ristic, 1974; Uilenberg et al., 2004). Enrichment, iso- muris Tyzzer and Weinman 1939, 143). lation and maintenance procedures, and methods of differentia- tion are essentially the same as those for genus Eperythrozoon.

List of species of the genus Haemobartonella 1. Mycoplasma haemomuris (Mayer 1921) Neimark, ­Johansson, Cells are coccoid to pleomorphic. Motility for this species Rikihisa and Tully 2002, 683VP (Bartonella muris Mayer 1921, has not been assessed. The morphology of infected erythro- 151; Bartonella muris ratti Regendanz and Kikuth 1928, 1578; cytes is altered, demonstrating a marked depression at the Haemobartonella muris Tyzzer and Weinman 1939, 143) site of Mycoplasma haemocanis attachment. This species has ha.e.mo.mu¢ris. Gr. neut. n. haema blood; L. masc. gen. n. not been grown on any artificial medium; therefore, nota- muris of the mouse; N.L. gen. n. haemomuris of mouse blood. ble biochemical parameters are not known. Pathogenic; causes hemolytic anemia in domestic dogs. Cells are coccoid and some display dense inclusion Transmission is vector-borne and mediated by the brown ­particles. Motility for this species has not been assessed. dog tick (Rhipicephalus sanguineus). The morphology of infected erythrocytes is altered, Source: observed in association with erythrocytes of ­demonstrating a marked depression at the site of Mycoplasma domestic dogs (Hoskins, 1991). haemomuris attachment. This species has not been grown DNA G+C content (mol%): Not determined. on any artificial medium; therefore, notable ­biochemical Type strain: not established. parameters are not known. Sequence accession no. (16S rRNA gene): AF197337. Opportunistic pathogen; causes anemia in splenecto- mized or otherwise immunosuppressed mice. Transmis- 3. Mycoplasma haemofelis (Clark 1942) Neimark, Johansson, sion is vector-borne and mediated by the rat louse (Polypax Rikihisa and Tully 2002, 683VP [Eperythrozoon felis Clark 1942, ­spinulosa). 16; Haemobartonella felis (Clark 1942) Flint and McKelvie Source: observed in association with erythrocytes of wild 1956, 240 and Kreier and Ristic 1984, 725] and captive mice, and hamsters. ha.e.mo.fe¢lis. Gr. neut. n. haema blood; L. fem. gen. n. felis DNA G+C content (mol%): not determined. of the cat; N.L. gen. n. haemofelis of cat blood. Type strain: not established. Sequence accession no. (16S rRNA gene): U82963. Cells are coccoid. Motility for this species has not been assessed. This species has not been grown on artificial medium; 2. Mycoplasma haemocanis (Kikuth 1928) Messick, Walker, therefore, notable biochemical parameters are not known. VP Raphael, Berent and Shi 2002, 697 [Bartonella canis Kikuth Pathogenic; causes hemolytic anemia in cats. The mode 1928, 1730; Haemobartonella (Bartonella) canis (Kikuth 1928) of transmission is percutaneous or oral; an insect vector has Tyzzer and Weinman 1939, 151; Kreier and Ristic 1984, not been identified although fleas have been implicated 726] (Woods et al., 2005). ha.e.mo.ca¢nis Gr. neut. n. haema blood; L. fem. gen. n. canis Tetracyclines and fluoroquinolones are effective thera- of the dog; N.L. gen. n. haemocanis of dog blood. peutic agents (Dowers et al., 2002; Tasker et al., 2006). Genus II. Haemobartonella 643

Source: observed in association with erythrocytes of in nomenclature. It is listed here merely because it has been domestic cats. incidentally cited as a species of Haemobartonella. DNA G+C content (mol%): 38.5 (genome sequence survey of strain OH; Berent and Messick, 2003). 1. “Haemobartonella procyoni” Frerichs and Holbrook 1971 Type strain: not established. Electron microscopy shows this epierythrocytic organism Sequence accession no. (16S rRNA gene): U88563. from a raccoon (Procyon lotor) is wall-less and its description The phylogenetic affiliations of the following proposed indicates it probably will prove to be a hemotropic myco- organism are unknown and its name does not have standing plasma (Frerichs and Holbrook, 1971).

References Man and Animals (edited by Weinman and Ristic). Academic Press, New York, pp. 387–472. Adler, S. and V. Ellenbogen. 1934. A note on two new blood parasites of Kreier, J.P. and M. Ristic. 1974. Genus IV. Haemobartonella Tyzzer and cattle: Eperythrozoon and Bartonella. J. Comp. Pathol. 47: 220–221. Weinman 1939, 143AL; Genus V. Eperythrozoon Schilling 1928, 1854AL. Berent, L.M. and J.B. Messick. 2003. Physical map and genome sequenc- In Bergey’s Manual of Determinative Bacteriology, 8th edn (edited ing survey of Mycoplasma haemofelis (Haemobartonella felis). Infect. by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. Immun. 71: 3657–3662. 910–914. Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal Kreier, J.P. and M. Ristic. 1984. Genus III. Haemobartonella; Genus IV. ­standards for description of new species of the class Mollicutes Eperythrozoon. In Bergey’s Manual of Systematic Bacteriology, vol. ­(division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. Brun-Hansen, H., H. Gronstol, H. Waldeland and B. Hoff. 1997. Eperyth- 724–729. rozoon ovis infection in a commercial flock of sheep. Zentralbl. Veteri- Li, X., X. Jia, D. Shi, Y. Xiao, S. Hu, M. Liu, Z. Yuan and D. Bi. 2008. narmed. B 44: 295–299. Continuous in vitro Cultivation of Mycoplasma suis. Acta Vet. Zootech. Clark, R. 1942. Eperythrozoon felis (sp. nov.) in a cat. J. Afr. Vet. Med. Sinica 38: 1142–1146. Assoc. 13: 15–16. Mason, R.W. and P. Statham. 1991. The determination of the level of Daddow, K.N. 1980. Culex annulirostris as a vector of Eperythrozoon ovis Eperythrozoon ovis parasitaemia in chronically infected sheep and its infection in sheep. Vet. Parasitol. 7: 313–317. significance to the spread of infection. Aust. Vet. J. 68: 115–116. Dowers, K.L., C. Olver, S.V. Radecki and M.R. Lappin. 2002. Use of Mayer, M. 1921. Über einige bakterienähnliche Parasiten der Erythrozyten enrofloxacin for treatment of large-form Haemobartonella felis in bei Menschen und Tieren. Arch. Schiffs Trop. Hyg. 25: 150–152. experimentally infected cats. J. Am. Vet. Med. Assoc. 221: 250–253. Messick, J.B., P.G. Walker, W. Raphael, L. Berent and X. Shi. 2002. ‘Can- Ewers, W.H. 1971. Eperythrozoon mariboi sp. nov. (Protophyta: order didatus Mycoplasma haemodidelphidis’ sp. nov., ‘Candidatus Myco- Richettsiales) a parasite of red blood cells of the flying fox Pteropus plasma haemolamae’ sp. nov. and Mycoplasma haemocanis comb. nov., macrotis epularius in New Guinea. Parasitology 63: 261–269. haemotrophic parasites from a naturally infected opossum (Didelphis Flint, J.C. and McKelvie D.H.. 1956. Feline infectious anemia-diagnosis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phyloge- and treatment. Proc. 92nd Ann. Meet. Am. Vet. Med. Assoc. 1955 netic and secondary structural relatedness of their 16S rRNA genes 240–242. to other mycoplasmas. Int. J. Syst. Evol. Microbiol. 52: 693–698. Frerichs, W.M. and A.A. Holbrook. 1971. Haemobartonella procyoni sp. n. Montes, A., D. Wolfe, E. Welles, J. Tyler and E. Tepe. 1994. Infertility in the raccoon, Procyon lotor. J. Parasitol. 57: 1309–1310. associated with Eperythrozoon wenyonii infection in a bull. J. Am. Vet. Groebel, K., K. Hoelzle, M.M. Wittenbrink, U. Ziegler and L.E. Hoelzle. Med. Assoc. 204: 261–263. 2009. Mycoplasma suis invades porcine erythrocytes. Infect. Immun. Neimark, H., K.E. Johansson, Y. Rikihisa and J.G. Tully. 2001. Pro- 77: 576–584. posal to transfer some members of the genera Haemobartonella and Gwaltney, S.M. and R.D. Oberst. 1994. Comparison of an improved Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candida- polymerase chain reaction protocol and the indirect hemaggluti- tus Mycoplasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, nation assay in the detection of Eperythrozoon suis infection. J. Vet. ‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma Diagn. Invest. 6 : 321–325. wenyonii’. Int. J. Syst. Evol. Microbiol. 51: 891–899. Hoelzle, L. 2008. Haemotrophic mycoplasmas: recent advances in Myco- Neimark, H., B. Hoff and M. Ganter. 2004. Mycoplasma ovis comb. nov. plasma suis. Vet. Microbiol. 130: 215–226. (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic Hoskins, J.D. 1991. Canine haemobartonellosis, canine hepatozoonosis, anaemia in sheep and goats. Int. J. Syst. Evol. Microbiol. 54: 365–371. and feline cytauxzoonosis. Vet. Clin. North Am. Small Anim. Pract. Neimark, H., W. Peters, B.L. Robinson and L.B. Stewart. 2005. Phylo- 21: 129–140. genetic analysis and description of Eperythrozoon coccoides, proposal Howard, G.W. 1975. The experimental transmission of Eperythrozoon ovis to transfer to the genus Mycoplasma as Mycoplasma coccoides comb. by mosquitoes. Parasitology 71: xxxiii. nov. and Request for an Opinion. Int. J. Syst. Evol. Microbiol. 55: Hoyte, H.M.D. 1962. Eperythrozoon teganodes sp. nov. (Rickettsiales), para- 1385–1391. sitic in cattle. Parasitology 52: 527–532. Neimark, H.C., K.E. Johansson, Y. Rikihisa and J.G. Tully. 2002. Revi- Hsu, F.S., M.C. Liu, S.M. Chou, J.F. Zachary and A.R. Smith. 1992. Eval- sion of haemotrophic Mycoplasma species names. Int. J. Syst. Evol. uation of an enzyme-linked immunosorbent assay for detection of Microbiol. 52: 683. Eperythrozoon suis antibodies in swine. Am. J. Vet. Res. 53: 352–354. Neitz, W.O., R.A. Alexander and P.J. de Toit. 1934. Eperythrozoon ovis (sp. Kikuth, W. 1928. Über Einen neuen Anämeerreger; Bartonella canis nov. nov.) infection in sheep. Onderstepoort J. Vet. Sci. 3: 263–274. spec. Klin. Wochenschr. 7: 1729–1730. Nikol’skii, S.N. and S.N. Slipchenko. 1969. Experiments in the trans- Kreier, J.P. and M. Ristic. 1963. Morphologic, antigenic, and pathogenic mission of Eperythrozoon ovis by the ticks H. plumbeum and Rh. bursa. characteristics of Eperythrozoon ovis and Eperythrozoon wenyoni. Am. Veterinariia (Russian) 5: 46. J. Vet. Res. 24: 488–500. Prullage, J.B., R.E. Williams and S.M. Gaafar. 1993. On the transmis- Kreier, J.P. and M. Ristic. 1968. Haemobartonellosis, eperythrozoono- sibility of Eperythrozoon suis by Stomoxys calcitrans and Aedes aegypti. Vet. sis, grahamellosis and ehrlichiosis. In Infectious Blood Diseases of Parasitol. 50: 125–135. 644 Family II. Incertae sedis

Regendanz, P. and W. Kikuth. 1928. Über Aktivierung labiler ­Infektionen Tyzzer, E.E. and D. Weinman. 1939. Haemobartonella n.g. (Bartonella olim duch Entmilzung (Piroplasma canis, Nuttalia brasiliensis, Bartonella pro parte) H. microti n. sp. of the field vole, Microtus pennsylvanicus. opossum, Spirochaeta didelphydis). Arch. f. Schiffs. U. Tropenhyg. 32: Am. J. Hyg. 30 : 141–157. 587–593. Uilenberg, G. 1967. [Eperythrozoon tuomii, n.sp. (Rickettsiales), the 3rd Schilling, V. 1928. Eperythrozoon coccoides, eine neue durch Splenektomie species of Eperythrozoon of cattle in Madagascar]. Rev. Elev. Med. Vet. aktivierbare Dauerinfektion der weissen Maus. Klin. Wochenschr. 7: Pays. Trop. 20 : 563–569. 1854–1855. Uilenberg, G., F. Thiaucourt and F. Jongejan. 2004. On molecular tax- Splitter, E.J. 1950. Eperythrozoon suis, the etiologic agent of icteroane- onomy: what is in a name? Exp. Appl. Acarol. 32: 301–312. mia–an anaplasmosis-like disease in swine. Am. J. Vet. Res. 11: Uilenberg, G., F. Thiaucourt and F. Jongejan. 2006. Mycoplasma and 324–329. Eperythrozoon (Mycoplasmataceae). Comments on a recent paper. Int. Sykes, J.E., N.L. Drazenovich, L.M. Ball and C.M. Leutenegger. 2007. J. Syst. Evol. Microbiol. 56: 13–14. Use of conventional and real-time polymerase chain reaction to Willi, B., F.S. Boretti, C. Baumgartner, S. Tasker, B. Wenger, V. Cattori, determine the epidemiology of hemoplasma infections in anemic M.L. Meli, C.E. Reusch, H. Lutz and R. Hofmann-Lehmann. 2006. and nonanemic cats. J. Vet. Intern. Med. 21: 685–693. Prevalence, risk factor analysis, and follow-up of infections caused Tasker, S., S.M. Caney, M.J. Day, R.S. Dean, C.R. Helps, T.G. by three feline hemoplasma species in cats in Switzerland. J. Clin. Knowles, P.J. Lait, M.D. Pinches and T.J. Gruffydd-Jones. 2006. Microbiol. 44: 961–969. Effect of chronic FIV infection, and efficacy of marbofloxacin Woods, J.E., M.M. Brewer, J.R. Hawley, N. Wisnewski and M.R. Lappin. treatment, on Mycoplasma haemofelis infection. Vet. Microbiol. 2005. Evaluation of experimental transmission of Candidatus Myco- 117: 169–179. plasma haemominutum and Mycoplasma haemofelis by Ctenocephalides Thurston, J.P. 1953. The chemotherapy of Eperythrozoon coccoides felis to cats. Am. J. Vet. Res. 66: 1008–1012. ­(Schilling 1928). Parasitology 43: 170–174. Zwart, D., P. Leeflang and C.J. van Vorstenbosch. 1969. Studies on Tuomi, J. and C.H. Von Bonsdorff. 1967. Ultrastructure of a microor- an Eperythrozoon associated with bovine thrombocytes. Zentralbl. ganism associated with bovine platelets. Experientia 23: 111–112. ­Bakteriol. [Orig.] 210: 82–105.

Order II. Entomoplasmatales Tully, Bové, Laigret and Whitcomb 1993, 381VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ro b e rt F. W h i t c o mb * En.to.mo.plas.ma.ta¢les. N.L. neut. n. Entomoplasma type genus of the order; -ales ending to denote an order: N.L. fem. pl. n. Entomoplasmatales the Entomoplasma order.

This order in the class Mollicutes has been assigned to a group Further descriptive information of nonhelical and helical mollicutes that are regularly associated The basis for the proposal for the order Entomoplasmatales (Tully with arthropod or plant hosts. The description of organisms in et al., 1993) was the distinctive phylogenetic and phenotypic the order is essentially the same as for the class. Two families are characteristics of culturable mollicutes regularly associated designated, Entomoplasmataceae for nonhelical mollicutes and with arthropods or plants. Members of the family Entomoplas- Spiroplasmataceae for helical ones. The order consists of four major mataceae are nonhelical mollicutes that differ in their choles- phylogenetic clades: the paraphyletic entomoplasmataceae clade, terol or serum requirements for growth. Nonhelical organisms which consists of the genera Entomoplasma and Mesoplasma; and with a strict requirement for cholesterol were placed in the the Apis, Citri–Chrysopicola–Mirum, and Ixodetis clades of the genus Entomoplasma (trivial name, entomoplasmas), whereas genus Spiroplasma. All cells are chemo-organotrophic, usually fer- nonhelical strains able to grow in a sterol-free medium supple- menting glucose through the phosphoenolpyruvate-dependent mented with PES were assigned to the genus Mesoplasma (trivial sugar transferase system. Arginine may be hydrolyzed, but urea is name, mesoplasmas). The proposal also included the transfer not. Cells may require sterol for growth. Nonhelical strains that of the family Spiroplasmataceae from the family Mycoplasmatales grow in serum-free media supplemented with polyoxyethylene to the family Entomoplasmatales. The helical organisms assigned sorbitan (PES) are currently assigned to the genus Mesoplasma. to the genus Spiroplasma were within the Spiroplasmataceae, and Temperature optimum for growth is usually 30–32°C, with a few genus and family descriptions of these organisms remained species able to grow at 37°C. Genome sizes range from 780 to as proposed previously (Skripal, 1983; Whitcomb and Tully, 2220 kbp by pulsed-field gel electrophoresis (PFGE), with DNA 1984). The order Entomoplasmatales is a phylogenetic sister to G+C contents ranging from 25 to 34 mol%. Like members of the the order Mycoplasmatales. These two orders together form a Mycoplasmatales, all organisms in this order are thought to utilize lineage with several unique properties, including the use of the UGA codon to encode tryptophan. UGA as a tryptophan codon rather than a stop codon. Type genus: Entomoplasma Tully, Bové, Laigret and Whitcomb 1993, 379VP. Taxonomic comments The genera Entomoplasma and Mesoplasma constitute a poly- phyletic sister lineage of the mycoides cluster of ­mycoplasmas that are eccentrically situated in the paraphyletic family Ento- *Deceased 21 December 2007. moplasmataceae (Gasparich et al., 2004). There is no current Family I. Entomoplasmataceae 645

­phylogenetic support for separation of Entomoplasma and (Tully et al., 1993), the species currently assigned to the genus Mesoplasma species based on neighbor-joining or maximum- Mesoplasma should most likely be transferred to the genus Ento- parsimony methods of 16S rRNA gene sequence similarity moplasma. Because the transfer would include its type species, analysis because they do not form coherent clusters, but are the genus Mesoplasma would then become illegitimate. More- instead intermixed in one paraphyletic group (Johansson over, Knight (2004) showed that the species formerly called and Pettersson, 2002; Tully et al., 1998). No DNA–DNA reas- Mesoplasma pleciae (Tully et al., 1994) is properly affiliated with sociation experiments have been performed nor is there any the genus Acholeplasma on undisputed grounds of 16S rRNA other polyphasic taxonomic basis to support the separation. gene sequence similarity and preferred use of UGG rather In particular, the growth requirement for sterols is not as pro- than UGA as the codon for tryptophan. Therefore, transfer of found a character as was initially believed and fails to justify the currently remaining members of genus Mesoplasma cannot these two species (Gasparich et al., 2004; Rose et al., 1993). be endorsed until similar analyses have been completed for all For these reasons, and because Entomoplasma has priority of those organisms (D.V. Volokhov, unpublished).

References phyletic cluster of arthropod-associated mollicutes to ordinal rank ­( Entomoplasmatales ord. nov.), with provision for familial rank to Gasparich, G.E., R.F. Whitcomb, D. Dodge, F.E. French, J. Glass and ­separate species with nonhelical morphology (Entomoplasmataceae D.L. Williamson. 2004. The genus Spiroplasma and its non-helical fam. nov.) from helical species (Spiroplasmataceae), and emended descendants: phylogenetic classification, correlation with phenotype descriptions of the order Mycoplasmatales, family Mycoplasmataceae. and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Micro- Int. J. Syst. Bacteriol. 43: 378–385. biol. 54: 893–918. Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, J.M. Johansson, K.E. and B. Pettersson. 2002. Taxonomy of Mollicutes. In Molec- Bove, P. Carle, D.L. Williamson and T.B. Clark. 1994. Taxonomic ular Biology and Pathogenicity of Mycoplasmas (edited by Razin and descriptions of eight new non-sterol-requiring Mollicutes assigned to Hermann). Kluwer Academic/Plenum Publishers, London, pp. 1–31. the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693. Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Achole- Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Williamson, F. Laigret, P. plasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene Carle, J.M. Bove, R.B. Henegar, N.M. Ellis, D.E. Dodge and J. Adams. sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. 1998. Entomoplasma freundtii sp. nov., a new species from a green tiger Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for beetle (Coleoptera: Cicindelidae). Int. J. Syst. Bacteriol. 48: 1197– measuring growth responses of Mollicutes to serum and polyoxyethyl- 1204. ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Whitcomb, R.F. and J.G. Tully. 1984. Family III. Spiroplasmataceae Skripal, I.G. 1983. Revival of the name Spiroplasmataceae fam. nov., nom. ­Skripal 1983, 408VP. Genus I. Spiroplasma Saglio, L’Hospital, Laflèche, rev., omitted from the 1980 Approved Lists of Bacterial Names. Int. Dupont, Bové, Tully and Freundt. In Bergey’s Manual of Systematic J. Syst. Bacteriol. 33: 408. Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised Baltimore, pp. 781–787. taxonomy of the class Mollicutes–proposed elevation of a mono-

Family I. Entomoplasmataceae Tully, Bové, Laigret and Whitcomb 1993, 380VP

* D a n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ro b e rt F. Wh i t c o mb En.to.mo.plas.ma.ta.ce¢ae. N.L. neut. n. Entomoplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Entomoplasmataceae the Entomoplasma family.

Cells are usually coccoid or occur as short, branched or Type genus: Entomoplasma Tully, Bové, Laigret and Whitcomb unbranched, pleomorphic, nonhelical filaments. Filterable 1993, 379VP. through membranes with a mean pore diameter of 220–450 nm. Cells lack a cell wall and are bounded only by a plasma Further descriptive information membrane. Nonmotile. Facultatively anaerobic. The tempera- All members of this paraphyletic family are nonhelical and ture range for growth varies from 10 to 37°C, with the optimum are regularly associated with arthropod or plant hosts. They usually at 30°C. The typical colony has a “fried-egg” appear- may require cholesterol or serum for growth, and most have ance. Chemo-organotrophic; acid is produced from glucose, an optimal growth temperature near 30°C. Separation of with evidence of a phosphoenolpyruvate-dependent sugar members of the genera Entomoplasma and Mesoplasma within transport system(s) in some members. Arginine and urea are the Entomoplasmataceae is based on the capacity of the Meso- not hydrolyzed. The organisms may require serum or choles- plasma species to grow in a serum-free or cholesterol-free terol for growth or may grow in serum-free media plus 0.04% medium supplemented with PES (Rose et al., 1993; Tully PES. The genome sizes range from 790 to 1140 kbp. et al., 1995), whereas Entomoplasma species have a growth DNA G+C content (mol%): 26–34. requirement for cholesterol. The family is derived from the Spiroplasma lineage and is most closely related to the Apis cluster of that group. The mycoides cluster of species in the genus Mycoplasma is related to this family and seems to have *Deceased 21 December 2007. evolved from it. 646 Family I. Entomoplasmataceae

Genus I. Entomoplasma Tully, Bové, Laigret and Whitcomb 1993, 379VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ro b e r t F. Wh i t c o mb * En.to.mo.plas¢ma. Gr. n. entomon insect; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Entomoplasma name intended to show association with insects. Cells are nonhelical and nonmotile, frequently pleomorphic ­viability after 7–10 d. Arginine hydrolysis and “film and spot” and range in size from 200 to 1200 nm in diameter. Some cells lipase reactions are rare among species described to date. Ento- exhibit short filamentous forms. Most species ferment glucose. moplasmas were shown to lack some key metabolic ­activities

Species possess the phosphoenolpyruvate-dependent sugar- found in other mollicutes, especially PPi-dependent phospho- phosphotransferase system. Organisms require serum or cho- fructokinase and dUTPase, and to possess uracil DNA glycosy- lesterol for growth. The temperature range for growth ranges lase activity. Although the latter pyrimidine enzymic activity from 10 to 32°C, with the optimum usually at 30–32°C. The distinguished Entomoplasma from Mesoplasma species, only two genome sizes range from 870 to 900 kbp (PFGE). All currently Entomoplasma species and three Mesoplasma species have been assigned species were isolated from insects or from plant sur- tested so far for these activities (Pollack et al., 1996). faces where they were presumably deposited by insects. Antisera to whole cell antigens of entomoplasmas have been DNA G+C content (mol%): 27–34. used extensively to provide specific identification to the species Type species: Entomoplasma ellychniae Tully, Rose, Hackett, level with a variety of serologic techniques, including growth Whitcomb, Carle, Bové, Colflesh and Williamson (Tully et al., inhibition, metabolism inhibition, and agar plate immunofluo- 1989) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Myco- rescence (Tully et al., 1989, 1990, 1998). There is no evidence plasma ellychniae Tully, Rose, Hackett, Whitcomb, Carle, Bové, for the pathogenicity of entomoplasmas to either plant or insect Colflesh and Williamson 1989, 288). hosts. Like other mollicutes, the entomoplasmas are resistant to 500 U/ml penicillin G. Further descriptive information Enrichment and isolation procedures Cells of these organisms vary from coccoid to pleomorphic forms exhibiting short, branching, nonhelical filaments. Round cells Flowers and other plant material should be cut in the field and are usually in the size range of 200–300 nm, but may be larger. placed in plastic bags without touching by hand. In the labora- Most strains were initially isolated in either M1D or SP-4 medium tory, plant materials are rinsed briefly in either SP-4 or M1D and all entomoplasmas grow well in SP-4 broth containing a sup- media (May et al., 2008). In both of these media, fetal bovine plement of 17% fetal bovine serum. Some strains are able to grow serum is a critical component for successful growth of these on media with reduced serum content. Most established species organisms (Hackett and Whitcomb, 1995; Tully, 1995). The rinse have an optimal growth temperature of 30°C, but some species medium is immediately decanted and passed through a sterile grow better in broth medium maintained at 23–25°C or at 32°C. membrane filter, usually of 450 nm porosity. The filtrate is then Colony growth on solid medium is best obtained on SP-4 medium passed through at least several tenfold dilutions in the selected incubated under anaerobic conditions at about 30°C. Under culture medium. The retentate may be frozen at −70°C for later these conditions, most species produce colonies with a classic use or for retesting. The cultures are incubated at 27–30°C and fried-egg appearance, although Entomoplasma freundtii is notable monitored by dark-field microscopy and/or by observing acidifi- for its granular colony morphology.­ cation of the medium. It is important to note that several non- All species show strong fermentation of glucose with produc- sterol-requiring Acholeplasma species have also been isolated from tion of acid and a reduction in medium pH (Table 140). Actively plant and insect material (Tully et al., 1994b). growing cultures in broth medium containing glucose may rap- Insect material, primarily from gut contents or hemolymph idly acidify the medium, causing partial or complete loss of obtained by dissection or by fine-pointed glass pipettes, should be added to small volumes of SP-4 or M1D medium and filtered through a 450 nm membrane filter. Serial tenfold dilutions of Table 140. Differential characteristics of species of the genus the filtrate should be incubated at 27–30°C and observed for a Entomoplasma a decrease in pH of the medium. After two to three serial pas- sages, the organisms should be purified by conventional filter- cloning techniques (Tully, 1983) and stocks of various clones and early passage isolates frozen for further identification pro- cedures (Whitcomb and Hackett, 1996).

Characteristic E. ellychniae E. freundtii E. lucivorax E. luminosum E. melaleucae E. somnilux Maintenance procedures Glucose fermentation + + + + + + Arginine hydrolysis − + − − − − Stock cultures of entomoplasmas can be maintained well in “Film and spots” − nd + + − − SP-4 and/or M1D broth medium containing about 17% fetal Hemadsorption of guinea − nd − + − − bovine serum. Most strains in the group can be adapted to pig red blood cells grow in a broth medium containing bovine serum. Stock cul- DNA G+C content (mol%) 27.7 34 27.4 28.8 27 27 tures in broth medium can be stored at −70°C for indefinite aSymbols: +, >85% positive; −, 0–15% positive; nd, not determined. periods. For optimum preservation, the organisms should be lyophilized as broth cultures in the early exponential phase of growth and the dried cultures should be sealed under vacuum *Deceased 21 December 2007. and stored at 4°C. Genus I. Entomoplasma 647

Differentiation of the genus Entomoplasma The paraphyletic relationship between the genera Ento- from other genera moplasma and Mesoplasma is currently an unresolved problem in the systematics of this genus. It is possible that these genera, sepa- Properties that partially fulfill criteria for assignment to the rated by the single criterion of sterol requirement, should be class Mollicutes (Brown et al., 2007) include absence of a cell combined into the single genus Entomoplasma. However, Knight wall, filterability, and the presence of conserved 16S rRNA gene (2004) showed that Mesoplasma pleciae (Tully et al., 1994b) should sequences. Aerobic or facultative anaerobic growth in artificial belong to the genus Acholeplasma based on 16S rRNA gene media and the necessity for sterols for growth exclude assign- sequence similarity and the preferred use of UGG rather than ment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, UGA as the codon for tryptophan. Therefore, transfer of the cur- Mesoplasma, or “Candidatus Phytoplasma”. Non-helical cellular rently remaining members of genus Mesoplasma to other genera morphology and regular association with arthropod or plant cannot be endorsed until similar analyses have been completed hosts support exclusion from the genera Spiroplasma or Myco- for all of those species (D.V. Volokhov, unpublished). plasma. The inability to hydrolyze urea excludes assignment to the genus Ureaplasma. However, the difficulty in assigning novel species to this genus is well demonstrated by the ear- Acknowledgements lier difficulties in establishing accurately the taxonomic status We thank Karl-Erik Johansson for helpful comments and sug- of these organisms (Tully et al., 1993). The availability of 16S gestions and Gail E. Gasparich for her landmark contributions rRNA gene sequence analyses was critical to the differentiation regarding the phylogenetics of the Entomoplasmatales. The major of these organisms from other mollicutes. Although isolates contributions to the foundation of this material by Joseph G. from vertebrates are very unlikely to be entomoplasmas, two Tully are gratefully acknowledged. bona fide Mycoplasma species, Mycoplasma iowae and Mycoplasma equigenitalium, have been isolated from plants [Grau et al., 1991; Further reading J.C. Vignault, J.M. Bové and J.G. Tully, unpublished (see ATCC 49192)]. Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas, vol. 5 (edited by Taxonomic comments Whitcomb and Tully). Academic Press, San Diego, pp. 1–31. The landmark studies of Weisburg et al. (1989), using 16S rRNA Tully, J.G. 1996. Mollicute–host interrelationships: current con- gene sequences of about 50 species of mollicutes, were critical cepts and diagnostic implications. In Molecular and Diagnos- in the resolution of certain taxonomic conflicts regarding the tic Procedures in Mycoplasmology, vol. 2 (edited by Tully and species that became Entomoplasma. The first entomoplasmas to Razin). Academic Press, San Diego, pp. 1–21. be recognized were serologically related isolates from the flow- ers of Melaleuca and Grevillea trees (McCoy et al., 1979). Others, Differentiation of the species of the genus found in a wide range of insect species (Tully et al., 1987), Entomoplasma included strain ELCN-1T from the hemolymph of the firefly The primary technique for differentiation of Entomoplasma beetle Ellychnia corrusca (Tully et al., 1989) and three serologi- species is 16S rRNA gene sequence comparisons, confirmed cally distinct strains isolated from gut contents of Pyractomena by serology (Brown et al., 2007). Nonhelical mollicutes that and Photinus beetles (Williamson et al., 1990). Although these belong to a known species isolated from arthropods or plants nonhelical, sterol-requiring mollicutes were initially placed in can be readily identified serologically provided that a battery the genus Mycoplasma, 16S rRNA gene sequence analysis clearly of potent antisera for classified species is available. Growth indicated that strain M1T, previously designated Mycoplasma inhibition tests, performed by placing paper discs saturated melaleucae, and strain ELCN-1T, previously designated Myco- with type-specific antisera on agar plates inoculated with plasma ellychniae, were most closely affiliated with the Spiroplasma the organism, are perhaps the most convenient and rapid lineage of helical organisms isolated primarily from arthro- serological technique to differentiate species (Clyde, 1983). pods. These findings prompted a proposal to reclassify the non- The agar plate immunofluorescence test is also a convenient helical mollicutes from arthropods and plants in a new order, and rapid means of mollicute species identification. In the Entomoplasmatales, and new family, Entomoplasmataceae, with absence of specific conjugated antiserum, an indirect immu- the genus Entomoplasma reserved for sterol-requiring ­species nofluorescence test can be performed with type-specific anti- (Tully et al., 1993). Strains M1T and ELCN-1T were renamed serum and a fluorescein-conjugated secondary antibody. The as Entomoplasma melaleucae and Entomoplasma ellychniae, respec- metabolism inhibition test (Taylor-Robinson, 1983) has also tively. Subsequent phylogenetic analysis of Mycoplasma freundtii, been applied to differentiation of Entomoplasma species (Tully later renamed Entomoplasma freundtii, confirmed the placement et al., 1998). (Tully et al., 1998).

List of species of the genus Entomoplasma

1. Entomoplasma ellychniae (Tully, Rose, Hackett, Whitcomb, el.lych.ni¢ae. N.L. n. Ellychnia a genus of firefly beetles; N.L. Carle, Bové, Colflesh and Williamson 1989) Tully, Bové, gen. n. ellychniae of Ellychnia, from which the organism was Laigret and Whitcomb 1993, 380VP (Mycoplasma ellychniae first isolated. Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and This is the type species of the genus Entomoplasma. Cells are Williamson 1989, 288) nonhelical, pleomorphic filaments, with some ­branching; 648 Family I. Entomoplasmataceae

small coccoid forms, ranging in diameter from 200 to 300 nm, No evidence of pathogenicity for insects or plants. also occur. Passage of broth cultures through 450 and 300 nm Source: first isolated from the gut of a firefly beetle (Photi- porosity membrane filters does not reduce viable cell ­numbers, nus pyralis); also isolated from a flower (Spirea ulmaria; whereas passage through 220 nm porosity reduces cell popula- C. Chastel, unpublished). tions by about 10%. Grows well in SP-4 medium with fetal DNA G+C content (mol%): 27.4 (Bd). bovine serum supplements. Does not grow well in horse serum- Type strain: PIPN-2, ATCC 49196, NCTC 11716. supplemented broth or agar media. Optimum temperature Sequence accession no. (16S rRNA gene): AF547212. for broth growth is 30°C; can grow at 18–32°C. Colonies incu- bated at 30°C under anaerobic conditions have a fried-egg 4. Entomoplasma luminosum (Williamson, Tully, Rose, ­Hackett, appearance. Does not hemadsorb guinea pig erythrocytes. Henegar, Carle, Bové, Colflesh and Whitcomb 1990) VP No evidence for pathogenicity for insects. Tully, Bové, Laigret and Whitcomb 1993, 380 (Mycoplasma Source: isolated from the hemolymph of the firefly beetle luminosum Williamson, Tully, Rose, Hackett, Henegar, Carle, Ellychniae corrusca. Bové, Colflesh and Whitcomb 1990, 163) DNA G+C content (mol%): 27.5 (Bd). lu.mi.no¢sum. L. neut. adj. luminosum luminous, emitting Type strain: ELCN-1, ATCC 43707, NCTC 11714. light, referring to the luminescence of the adult host from Sequence accession no. (16S rRNA gene): M24292. which the organism was isolated. 2. Entomoplasma freundtii Tully, Whitcomb, Hackett, Williamson, Cells are pleomorphic and coccoidal or subcoccoidal Laigret, Carle, Bové, Henegar, Ellis, Dodge and Adams 1998, with a diameter of 200–300 nm. Cells also occur as short, 1203VP branched or unbranched filaments. The organisms are read- ily filterable through membranes with mean pore diameters freund¢ti.i. N.L. masc. gen. n. freundtii of Freundt, named of 450, 300, and 220 nm, but do not pass 100 nm porosity after Eyvind Freundt, a Danish pioneer in the taxonomy and membranes. The temperature range for growth is 10–32°C, classification of mollicutes. with an optimum at 32°C. Nonmotile. Colonies under anaer- Cells are predominantly coccoid in shape, ranging from obic conditions have a fried-egg appearance. The organism 300 to 1200 nm in diameter. Organisms are readily filterable grows well in SP-4 broth medium or other media containing through membranes with mean pore diameters of 450, 300, horse serum supplements. Colonies hemadsorb guinea pig and 220 nm; more than 90% of viable cells in broth culture erythrocytes. are able to pass 220 nm porosity membranes. The tempera- No evidence of pathogenicity for insects. ture range for growth is 10–32°C, with an optimum at 30°C. Source: isolated from the gut of the firefly beetle (Photinus Colonies under anaerobic conditions are granular and fre- marginata). quently exhibit multiple satellite forms although the organ- DNA G+C content (mol%): 28.8 (Bd). ism is considered nonmotile. The organism grows well in Type strain: PIMN-1, ATCC 49195, NCTC 11717. SP-4 broth medium or other media containing horse serum Sequence accession no. (16S rRNA gene): AY155670. supplements. No evidence for pathogenicity for insects. 5. Entomoplasma melaleucae (Tully, Rose, McCoy, Carle, Bové, Source: isolated from the gut contents of a green tiger bee- Whitcomb and Weisburg 1990) Tully, Bové, Laigret and Whit- VP tle (Coleoptera: Cicindelidae). comb 1993, 380 (Mycoplasma melaleucae Tully, Rose, McCoy, DNA G+C content (mol%): 34.1 (Bd). Carle, Bové, Whitcomb and Weisburg 1990, 146) Type strain: BARC 318, ATCC 51999. me la.leu¢cae. N.L. n. Melaleuca a genus of tropical trees hav- Sequence accession no. (16S rRNA gene): AF036954. ing white flowers with sweet fragrance; N.L. gen. n. melaleucae of Melaleuca, the plant from which the type strain was iso- 3. Entomoplasma lucivorax (Williamson, Tully, Rose, Hackett, lated. Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Cells are pleomorphic and coccoidal or subcoccoidal, with Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma lucivo- few filamentous forms. Coccoidal forms have mean diameters rax Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, of 250–300 nm. Cells are readily filterable through 450 and Colflesh and Whitcomb 1990, 164) 300 nm porosity membrane filters, with few cells passing 220 lu.ci.vo¢rax. L. fem. n. lux lucis light; L. neut. adj. vorax glut- nm porosity membranes. The temperature range for growth tonous, devouring; N.L. neut. adj. lucivorax light devouring, is 10–30°C, with an optimum at about 23°C. Nonmotile. Col- referring to the predacious habit of the host insect, which onies under anaerobic conditions at 23–30°C display a fried- preys on other luminescent firefly species. egg appearance. Grows well in SP-4 broth or in modified Cells are either pleomorphic coccoidal or subcoccoidal, Edward medium containing fetal bovine serum. The organ- with a diameter of 200–300 nm, or are short, branched or ism does not grow well in horse serum-based broth medium. unbranched filaments. Cells are readily filterable through Agar colonies do not adsorb guinea pig erythrocytes. membrane filters with mean pore diameters of 450, 300, and No evidence of pathogenicity for insects or plants. 220 nm, but do not pass 100 nm porosity membranes. Opti- Source: isolated from flower surfaces of a subtropical plant, mum temperature for growth is 30°C; can grow at 10–32°C. Melaleuca quinquenervia, in south Florida. Related strains Nonmotile. Colonies under anaerobic conditions usually have have been isolated from flowers of other subtropical trees a fried-egg appearance. Grows well in SP-4 broth medium or in Florida, Melaleuca decora and Grevillea robusta (silk oak), other media containing horse serum supplements. Colonies and from an anthophorine bee (Xylocopa micans) in the same do not hemadsorb guinea pig erythrocytes. geographic area. Genus II. Mesoplasma 649

DNA G+C content (mol%): 27.0 (Bd). Cells are pleomorphic and coccoidal or subcoccoidal, with Type strain: M1, ATCC 49191, NCTC 11715. a diameter of 200–300 nm; also occur as short, branched or Sequence accession nos (16S rRNA gene): M24478, AY345990. unbranched filaments. Readily filterable through membranes Further comment: the 16S rRNA gene sequence is more sim- with mean pore diameters of 450, 300, and 220 nm. The tem- ilar to that of members of genus Mesoplasma than to others in perature range for growth is 10–32°C, with ­optimum growth the genus Entomoplasma. at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. The organ- 6. Entomoplasma somnilux (Williamson, Tully, Rose, Hackett, ism grows well in SP-4 broth medium or other media con- Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, taining horse serum supplements. Colonies do not adsorb Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma guinea pig erythrocytes. ­somnilux Williamson, Tully, Rose, Hackett, Henegar, Carle, No evidence of pathogenicity for insects. Bové, Colflesh and Whitcomb 1990, 163) Source: isolated from a pupal gut of the firefly beetle (Pyra- som.ni¢lux. L. masc. n. somnus sleep; L. fem. n. lux light; N.L. ctomena angulata). n. somnilux intended to mean sleeping light, referring to the DNA G+C content (mol%): 27.4 (Bd). quiescent pupal stage of the host from which the organism Type strain: PYAN-1, ATCC 49194, NCTC 11719. was isolated, which precedes the luminescent adult stage. Sequence accession no. (16S rRNA gene): AY157871.

Genus II. Mesoplasma Tully, Bové, Laigret and Whitcomb 1993, 380VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ro b e r t F. Wh i t c o mb * Me.so.plas¢ma. Gr. adj. mesos middle; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Mesoplasma middle form, name intended to denote a middle position with respect to sterol or cholesterol requirement. Cells are nonhelical and nonmotile, generally coccoid or short bovine serum, or in broth medium containing a 1% bovine filamentous forms. Coccoid cells are usually 220–300 nm in dia­ serum fraction supplement (Tully, 1984; Tully et al., 1994a). All meter, but some cells in some species can be as large as 400–500 nm. species show strong fermentation of glucose with acid produc- Most strains ferment glucose and most, but not all, lack the ability tion (Table 141), with a rapid decline in pH of the medium and to hydrolyze arginine. Species possess the phosphoenolpyruvate- loss of viability. Arginine hydrolysis has been observed only with dependent sugar-phosphotransferase system. Neither serum nor the type strain (PUPA-2T) of Mesoplasma photuris. cholesterol is required for growth, but strains show sustained Antisera directed against whole-cell antigens of filter-cloned growth in a serum-free or cholesterol-free medium when the mesoplasmas have been used extensively to establish species medium is supplemented with 0.04% PES. The optimum temper- and to provide species identifications. There is no evidence of ature for growth is usually near 28–32°C, with some strains able to pathogenicity of any currently established species in the genus grow well at temperatures as low as 23°C or as high as 37°C. for either an insect or plant host. Mesoplasmas are resistant to Genome sizes range from 825 to 930 kbp (PFGE). 500 U/ml penicillin. DNA G+C content (mol%): 26–32. Type species: Mesoplasma florum (McCoy, Basham, Tully, Rose, Enrichment, isolation, and maintenance procedures Carle and Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, The culture media and procedures for isolation and mainte- 380VP (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle nance of entomoplasmas from plant and insect sources can also and Bové 1984, 14). be effectively applied for mesoplasmas. Further descriptive information Differentiation of the genus Mesoplasma Cells are predominantly coccoid in the exponential phase of from other genera growth when examined by dark-field microscopy. Cells from Properties that fulfill criteria for assignment to this genus are broth cultures examined by transmission electron microscopy the same as those for the genus Entomoplasma, with the excep- are also coccoid, with individual cells usually 220–500 nm in tion that the genus Mesoplasma is currently reserved for species diameter and clearly defined by a single cytoplasmic mem- that are able to grow in serum-free medium supplemented with brane. Colony growth is best obtained on SP-4 agar medium. PES (Tully et al., 1993). Plates incubated under anaerobic conditions at about 30°C usually display characteristic fried-egg type colonies after 5–7 Taxonomic comments d incubation. The existence of a flora of nonhelical, wall-less prokaryotes Several mesoplasmas lack certain key metabolic activities associated with arthropod or plant hosts was first documented found in other mollicutes, especially PPi-dependent phospho- by T.B. Clark, S. Eden-Green, and R.E. McCoy and colleagues. fructokinase, dUTPase, and uracil DNA glycosylase activity Some of the plant isolates were clearly related to previously (Pollack et al., 1996). Most mesoplasmas were isolated in M1D described Acholeplasma species, such as Acholeplasma oculi (Eden- medium containing 15% fetal bovine serum (Whitcomb, 1983), Green and Tully, 1979), whereas others were established as novel but adapt well to growth in SP-4 broth containing 15–17% fetal Acholeplasma species, able to grow well in broth media without any cholesterol, serum, or fatty acid supplements. However, a *Deceased 21 December 2007. significant group of other similarly derived strains were able to 650 Family I. Entomoplasmataceae

Table 141. Differential characteristics of species of the genus Mesoplasma a

Characteristic M. florum M. chauliocola M. coleopterae M. corruscae M. entomophilum M. grammopterae M. lactucae M. photuris M. seiffertii M. syrphidae M. tabanidae Glucose fermentation + + + + + + + + + + + Arginine hydrolysis − − − − − − − + − − − Hemadsorption of guinea pig red blood cells − + − + + − + − + + − DNA G+C content (mol%) 27.3 28.3 27.7 26.4 30 29.1 30 28.8 30 27.6 28.3 aSymbols: +, >85% positive; −, 0–15% positive. grow in serum-free or cholesterol-free media only when small ­separated by the single criterion of sterol requirement, should amounts of PES were added to the medium. Because these be combined into the single genus Entomoplasma. However, strains grew in the absence of cholesterol or serum, several of Knight (2004) showed that Mesoplasma pleciae (Tully et al., them were initially described as Acholeplasma species, including 1994a) should belong to the genus Acholeplasma based on 16S Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophi- rRNA gene sequence similarity and the preferred use of UGG lum (Tully et al., 1988), and Acholeplasma seiffertii (Bonnet et al., rather than UGA as the codon for tryptophan. Therefore, trans- 1991). Although the growth response to PES in serum-free or fer of the currently remaining members of the genus Meso- cholesterol-free media suggested that there were fundamental plasma to other genera cannot be endorsed until similar analyses differences between such mollicutes and classic acholeplasmas, have been completed for all of those species (D.V. Volokhov, conclusive taxonomic evidence was lacking. The subsequent unpublished). analysis of 16S rRNA gene sequences by Weisburg et al. (1989) showed that the PES-requiring organisms were closely related Acknowledgements to the spiroplasma group of mollicutes and were phylogeneti- We thank Karl-Erik Johansson for helpful comments and sug- cally distant from acholeplasmas. On the basis of these findings gestions and Gail E. Gasparich for her landmark contributions and additional phylogenetic data, a proposal was made that the regarding the phylogenetics of the Entomoplasmatales. The major plant- and insect-derived mollicutes with growth responses to contributions to the foundation of this material by Joseph G. PES in serum-free or cholesterol-free media would be assigned Tully are gratefully acknowledged. to a new family, Entomoplasmataceae, and a new genus, Mesoplasma Further reading (Tully et al., 1993). Three of the plant-derived strains previously described as Acholeplasma species (Acholeplasma f­lorum, Achole- Tully, J.G. 1989. Class Mollicutes: new perspectives from plant plasma entomophilum, and Acholeplasma seiffertii) were transferred and arthropod studies. In The Mycoplasmas, vol. 5 (edited by to the genus Mesoplasma, with retention of their species epithets. Whitcomb and Tully). Academic Press, San Diego, pp. 1–31. A single plant-derived strain that had previously been described Tully, J.G. 1996. Mollicute-host interrelationships: current con- as Mycoplasma lactucae, and later found to grow in serum-free or cepts and diagnostic implications. In Molecular and Diagnos- cholesterol-free media supplemented with PES, was renamed tic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Mesoplasma lactucae. Later, eight novel Mesoplasma species were Razin). Academic Press, San Diego, pp. 1–21. described (Tully et al., 1994a). The paraphyletic relationship between the genera Ento- Differentiation of the species of the genus Mesoplasma moplasma and Mesoplasma is a currently unresolved problem in The techniques for differentiation of Mesoplasma species are the the systematics of this genus. It is possible that these genera, same as those for genus Entomoplasma.

List of species of the genus Mesoplasma 1. Mesoplasma florum (McCoy, Basham, Tully, Rose, Carle and incubation at 37°C. Colonies on agar do not hemadsorb Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, 380VP guinea pig erythrocytes. (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and The 16S rRNA gene sequence is identical to that of Meso- Bové 1984, 14) plasma entomophilum (GenBank accession no. AF305693), flo¢rum. L. gen. pl. n. florum of flowers, indicating the recov- but antiserum against Mesoplasma florum did not inhibit ery site of the organism. growth of Mesoplasma entomophilum or label the surfaces of This is the type species of the genus. Cells are oval or coc- Mesoplasma entomophilum colonies on agar (Tully et al., 1988). coid. The organism is readily filterable through membranes There are additional phenotypic distinctions between the with mean pore diameters of 450, 300, and 220 nm, but does two species. not pass a membrane with 100 nm porosity. Temperature No evidence of pathogenicity for plants or insects. range for growth is 18–37°C, with an optimum at 28–30°C. Source: first isolated from surface of flowers on a Colonies on agar medium containing horse serum supple- lemon tree (Citrus limon) in Florida, with subsequent ments have a typical fried-egg appearance after anaerobic ­isolations from floral surfaces of grapefruit (Citrus Genus II. Mesoplasma 651

­paradisi) and ­powderpuff trees (Albizia julibrissin) in ­Temperature range for growth is 10–32°C, with an optimum Florida (McCoy et al., 1979). Also isolated from a variety of 30°C. Nonmotile. Colonies incubated anaerobically at of plants and from the gut tissues of numerous species of 30°C usually have a fried-egg appearance. Colonies hemad- insects (Clark et al., 1986; Tully et al., 1990; Whitcomb sorb guinea pig erythrocytes. et al., 1982). No evidence of pathogenicity for plants or insects. DNA G+C content (mol%): 27.3 (Bd, whole genome Source: original isolation was from the gut of an adult sequence). ­fireflyEllychnia ( corrusca).

Type strain: L1, ATCC 33453, NCTC 11704. DNA G+C content (mol%): 26.4 (Bd, Tm, HPLC). Sequence accession nos: AF300327 (16S rRNA gene), Type strain: ELCA-2, ATCC 49579. NC_006055 (strain L1T genome sequence). Sequence accession no. (16S rRNA gene): AY168929. 2. Mesoplasma chauliocola Tully, Whitcomb, Hackett, Rose, 5. Mesoplasma entomophilum (Tully, Rose, Carle, Bové, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP Hackett and Whitcomb 1988) Tully, Bové, Laigret and ­Whitcomb 1993, 380VP (Acholeplasma entomophilum Tully, chau.li.o¢co.la. N.L. n. chaulio first part of the genus name Rose, Carle, Bové, Hackett and Whitcomb 1988, 166) of goldenrod beetle (Chauliognathus); L. suff. -cola (from L. masc. or fem. n. incola) inhabitant; N.L. masc. n. chauliocola en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. inhabitant of the goldenrod beetle. philum­ (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. entomophilum insect-loving. Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells are readily filterable through Cells are pleomorphic, but primarily coccoid, ranging membranes with mean pore diameters of 450, 300, and from 300 to 500 nm in diameter. Cells are readily filterable 220 nm, with a small number of cells able to pass through through 220 nm porosity membrane filters. The tempera- 100 nm porosity filters. Temperature range for growth is ture range for growth is 23–32°C, with an optimum at 30°C. 10–37°C, with an optimum of 32–37°C. Nonmotile. Colo- Nonmotile. Colonies incubated under anaerobic condi- nies incubated anaerobically at 32–37°C show fried-egg tions at 30°C usually have a fried-egg appearance. Colonies morphology. Colonies hemadsorb guinea pig erythrocytes. hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. The 16S rRNA gene sequence is identical to that of Source: originally isolated from gut fluid of an adult gold- Mesoplasma­ florum (GenBank accession no. AF300327), but enrod soldier beetle (Chauliognathus pennsylvanicus). antiserum against Mesoplasma florum did not inhibit growth of Mesoplasma entomophilum or label the surfaces of Meso- DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC). Type strain: CHPA-2, ATCC 49578. plasma entomophilum colonies on agar (Tully et al., 1988). Sequence accession no. (16S rRNA gene): AY166704. There are additional phenotypic distinctions between the two species. 3. Mesoplasma coleopterae Tully, Whitcomb, Hackett, Rose, No evidence of pathogenicity for plants or insects. VP Henegar, Bové, Carle, Williamson and Clark 1994a, 692 Source: original isolation was from the gut contents of a co.le.op.te¢rae. N.L. fem. gen. n. coleopterae of Coleoptera, tabanid fly Tabanus( catenatus). Also isolated from a variety referring to the order of insects (Coleoptera) from which of other species of insects. the organism was first isolated. DNA G+C content (mol%): 30 (Bd). Cells are primarily coccoid, ranging in diameter from Type strain: TAC, ATCC 43706, NCTC 11713. 300 to 500 nm. Organisms are readily filterable through Sequence accession no. (16S rRNA gene): AF305693. membranes with mean pore diameters of 450, 300, and 6. Mesoplasma grammopterae Tully, Whitcomb, Hackett, Rose, 220 nm. Temperature range for growth is 10–37°C, with an Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP optimum of 30–37°C. Nonmotile. Colonies incubated anaerobically at 30°C usually have a fried-egg appearance. gram.mop.te¢rae. N.L. fem. gen. n. grammopterae of Gram- Agar colonies do not hemadsorb guinea pig erythrocytes. moptera, referring to the genus of beetle (Grammoptera) from No evidence of pathogenicity for plants or insects. which the organism was first isolated. Source: original isolation was from the gut of an adult Cells are primarily coccoid, ranging in diameter from ­soldier beetle (Chauliognathus sp.). 300 to 500 nm. Cells are readily filterable through mem- brane filters with mean pore diameters of 450, 300, and DNA G+C content (mol%): 27.7 (Bd, Tm, HPLC). Type strain: BARC 779, ATCC 49583. 220 nm. Temperature range for growth is 10–37°C, with an Sequence accession no. (16S rRNA gene): DQ514605 (partial optimum at 30°C. Nonmotile. Colonies incubated under sequence). anaerobic conditions at 30°C have a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes. 4. Mesoplasma corruscae Tully, Whitcomb, Hackett, Rose, No evidence of pathogenicity for plants or insects. Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP Source: original isolation was from the gut contents of an cor.rus¢cae. N.L. fem. gen. n. corruscae of corrusca, refer- adult long-horned beetle (Grammoptera sp.). Other isola- ring to the species of firefly beetle (Ellychnia corrusca) from tions were made from adult soldier beetle (Cantharidae sp.) which the organism was first isolated. and from an adult mining bee (Andrena sp.).

Cells are primarily coccoid, ranging in diameter from DNA G+C content (mol%): 29.1 (Bd, Tm, HPLC). 300 to 500 nm. Cells are readily filterable through mem- Type strain: GRUA-1, ATCC 49580. branes with mean pore diameters of 450, 300, and 220 nm. Sequence accession no. (16S rRNA gene): AY174170. 652 Family I. Entomoplasmataceae

7. Mesoplasma lactucae (Rose, Kocka, Somerson, Tully, 220 nm. Temperature range for growth is 20–35°C, ­Whitcomb, Carle, Bové, Colflesh and Williamson 1990) with optimum at about 28–30°C. Nonmotile. Colonies Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma incubated under anaerobic conditions at 30°C have a lactucae Rose, Kocka, Somerson, Tully, Whitcomb, Carle, fried-egg appearance. Colonies hemadsorb guinea pig Bové, Colflesh and Williamson 1990, 141) erythrocytes. lac.tu¢cae. L. fem. n. lactuca lettuce; L. gen. n. lactucae of Three insect isolates of Mesoplasma seiffertii, two from lettuce, referring to the plant from which the organism was ­mosquitoes and one from a horse fly, were compared to T first isolated. strain F7 of plant origin. High relatedness values of 78–98% DNA–DNA reassociation under high stringency conditions Cells are primarily coccoid, ranging in size from 300 to were obtained (Gros et al., 1996). 500 nm in diameter, with only occasional short, nonhelical, No evidence of pathogenicity for plants or insects. pleomorphic filaments. Cells are readily filterable through Source: first isolated from floral surfaces of a sweet orange membrane filters with mean pore diameters of 450, 300, tree (Citrus sinensis) and from wild angelica (Angelica sylves- and 220 nm, and a few cells are able to pass 100 nm poros- tris). Also isolated from insects. ity membranes. Temperature range for growth is 18–37°C, DNA G+C content (mol%): 30 (Bd). with optimal growth at 30°C. Nonmotile. Colonies incu- Type strain: F7, ATCC 49495. bated under anaerobic conditions at 30°C have a fried-egg Sequence accession no. (16S rRNA gene): L12056. appearance. Colonies hemadsorb guinea pig erythrocytes. No evidence of pathogenicity for plants or insects. Source: original isolation was from lettuce (Lactuca 10. Mesoplasma syrphidae Tully, Whitcomb, Hackett, Rose, VP sativa). Henegar, Bové, Carle, Williamson and Clark 1994a, 691 DNA G+C content (mol%): 30 (Bd). syr.phi¢dae. N.L. fem. gen. n. syrphidae of a syrphid, refer- Type strain: 831-C4, ATCC 49193, NCTC 11718. ring to the syrphid fly family (Syrphidae), from which the Sequence accession no. (16S rRNA gene): AF303132. Has been organism was first isolated. reported to possess three rRNA operons (Grau, 1991). Cells are primarily coccoid, ranging in size from 300 to 8. Mesoplasma photuris Tully, Whitcomb, Hackett, Rose, 500 nm in diameter. Cells readily pass membrane filters ­Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP with mean pore diameters of 450, 300, and 220 nm. Tem- perature range for growth is 10–32°C, with optimum at pho.tu¢ris. N.L. gen. n. photuris of Photuris, referring to the 23–25°C. Nonmotile. Colonies incubated under anaerobic genus of firefly beetle (Photuris sp.) from which the organ- conditions at 23–25°C have a fried-egg appearance. Colo- ism was first isolated. nies hemadsorb guinea pig erythrocytes. Cells are primarily coccoid, ranging in diameter from No evidence of pathogenicity for insects. 300 to 500 nm. Readily filterable through membrane filters Source: original isolation was from the gut of an adult with mean pore diameters of 450, 300, and 220 nm. Tem- syrphid fly (Diptera: Syrphidae). Similar strains have been perature range for growth is 10–32°C, with optimum at isolated from a bumblebee (Bombus sp.) and a skipper 30°C. Nonmotile. Colonies incubated under anaerobic con- ­(Lepidoptera: Hesperiidae). ditions at 30°C have a fried-egg appearance. Colonies do DNA G+C content (mol%): 27.6 (Bd, Tm, HPLC). not hemadsorb guinea pig erythrocytes. Type strain: YJS, ATCC 51578. No evidence of pathogenicity for plants or insects. Sequence accession no. (16S rRNA gene): AY231458. Source: original isolation was from gut fluids of larval and adult firefliesPhoturis ( lucicrescens and other Photuris spp.). 11. Mesoplasma tabanidae Tully, Whitcomb, Hackett, Rose, One isolate (BARC 1976) was obtained by F.E. French from Henegar, Bové, Carle, Williamson and Clark 1994a, 692VP the gut of a horse fly Tabanus( americanus). ta.ba.ni.dae. N.L. fem. gen. n. tabanidae of a tabanid, refer- DNA G+C content (mol%): 28.8 (Bd, Tm, HPLC). Type strain: PUPA-2, ATCC 49581. ring to the horse fly family (Tabanidae), the host from Sequence accession no. (16S rRNA gene): AY177627. which the organism was first isolated. Cells are primarily coccoid, ranging in size from 300 9. Mesoplasma seiffertii (Bonnet, Saillard, Vignault, Garnier, to 500 nm in diameter. Cells readily pass membrane fil- Carle, Bové, Rose, Tully and Whitcomb 1991) Tully, Bové, ters with mean pore diameters of 450, 300, and 220 nm. Laigret and Whitcomb 1993, 380VP (Acholeplasma seiffertii Temperature range for growth is 10–37°C, with optimum Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully at 37°C. Nonmotile. Colonies incubated under anaerobic and Whitcomb 1991, 48) conditions at 37°C display a fried-egg appearance. Colonies seif.fer¢ti.i. N.L. masc. gen. n. seiffertii of Seiffert, in honor do not hemadsorb guinea pig erythrocytes. of Gustav Seiffert, a German microbiologist who performed No evidence of pathogenicity for insects. pioneering studies on mollicutes that occur in soil and com- Source: original isolation was from the gut of an adult post and do not require sterols for growth. horse fly Tabanus( abactor).

Cells are primarily coccoid, ranging in diameter DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC). from 300 to 500 nm. Cells are readily filterable through Type strain: BARC 857, ATCC 49584. membranes with mean pore diameters of 450, 300, and Sequence accession no. (16S rRNA gene): AY187288. Genus II. Mesoplasma 653

References Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene ­sorbitan growth requirements of mollicutes. In Molecular and Diag- Bonnet, F., C. Saillard, J.C. Vignault, M. Garnier, P. Carle, J.M. Bové, D.L. nostic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Rose, J.G. Tully and R.F. Whitcomb. 1991. Acholeplasma seiffertii sp. nov., Tully). Academic Press, San Diego, pp. 381–389. a mollicute from plant surfaces. Int. J. Syst. Bacteriol. 41: 45–49. Tully, J.G., D.L. Rose, R.F. Whitcomb, K.J. Hackett, T.B. Clark, Brown, D.R., R.F. Whitcomb and J.M. Bradbury. 2007. Revised minimal R.B. Henegar, E. Clark, P. Carle and J.M. Bové. 1987. Characteriza- standards for description of new species of the class Mollicutes (divi- tion of some new insect-derived acholeplasmas. Isr. J. Med. Sci. 23: sion Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. 699–703. Clark, T.B., J.G. Tully, D.L. Rose, R. Henegar and R.F. Whitcomb. Tully, J.G., D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett and R.F. Whit- 1986. Acholeplasmas and similar nonsterol-requiring mollicutes comb. 1988. Acholeplasma entomophilum sp. nov. from gut contents of from insects: missing link in microbial ecology. Curr. Microbiol. 13: a wide-range of host insects. Int. J. Syst. Bacteriol. 38: 164–167. 11–16. Tully, J.G., D.L. Rose, K.J. Hackett, R.F. Whitcomb, P. Carle, J.M. Bové, Clyde, W.A., Jr. 1983. Growth inhibition tests. In Methods in Mycoplas- D.E. Colflesh and D.L. Williamson. 1989. Mycoplasma ellychniae sp. mology, vol. 1 (edited by Razin and Tully). Academic Press, New nov., a sterol-requiring mollicute from the firefly beetle Ellychnia cor- York, pp. 405–410. rusca. Int. J. Syst. Bacteriol. 39: 284–289. Eden-Green, S.J. and J.G. Tully. 1979. Isolation of Acholeplasma spp. Tully, J.G., D.L. Rose, R.E. McCoy, P. Carle, J.M. Bové, R.F. Whitcomb from coconut palms affected by lethal yellowing disease in Jamaica. and W.G. Weisburg. 1990. Mycoplasma melaleucae sp. nov., a sterol- Curr. Microbiol. 2: 311–316. requiring mollicute from flowers of several tropical plants. Int. J. Syst. Grau, O. 1991. Analyse des gènes ribosomiques des mollicutes, appli- Bacteriol. 40: 143–147. cation à l’identification d’un mollicute non classé et conséquences Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised tax- taxonomiques [thesis]. Bordeaux, France. onomy of the class Mollicutes - proposed elevation of a monophyletic Grau, O., F. Laigret, P. Carle, J.G. Tully, D.L. Rose and J.M. Bové. 1991. cluster of arthropod-associated mollicutes to ordinal rank (Ento- Identification of a plant-derived mollicute as a strain of an avian moplasmatales ord. nov.), with provision for familial rank to separate pathogen, Mycoplasma iowae, and its implications for mollicute tax- species with nonhelical morphology (Entomoplasmataceae fam. nov.) onomy. Int. J. Syst. Bacteriol. 41: 473–478. from helical species (Spiroplasmataceae), and emended descriptions Gros, O., C. Saillard, C. Helias, F. LeGoff, M. Marjolet, J.M. Bové and of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bac- C. Chastel. 1996. Serological and molecular characterization of Meso- teriol. 43: 378–385. plasma seiffertii strains isolated from hematophagous dipterans in Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, J.M. France. Int. J. Syst. Bacteriol. 46: 112–115. Bové, P. Carle, D.L. Williamson and T.B. Clark. 1994a. Taxonomic Hackett, K.J. and R.F. Whitcomb. 1995. Cultivation of spiroplasmas descriptions of eight new non-sterol-requiring Mollicutes assigned to in undefined and defined media. In Molecular and Diagnostic the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693. Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Tully, J.G., R.F. Whitcomb, D.L. Rose, J.M. Bové, P. Carle, N.L. Somerson, ­Academic Press, San Diego, pp. 41–53. D.L. Williamson and S. Edengreen. 1994b. Acholeplasma brassicae sp. Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Achole- nov. and Acholeplasma palmae sp. nov., two ­non-sterol-requiring molli- plasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene cutes from plant surfaces. Int. J. Syst. Bacteriol. 44: 680–684. sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. Tully, J.G., D.L. Rose, C.E. Yunker, P. Carle, J.M. Bové, D.L. Williamson May, M., R.F. Whitcomb and D.R. Brown. 2008. Mycoplasma and related and R.F. Whitcomb. 1995. Spiroplasma ixodetis sp. nov., a new species organisms. In Practical Handbook of Microbiology (edited by Gold- from Ixodes pacificus ticks collected in Oregon. Int. J. Syst. Bacteriol. man and Green). CRC Press, Boca Raton, pp. 467–491. 45: 23–28. McCoy, R.E., D.S. Williams and D.L. Thomas. 1979. Isolation of myco- Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Williamson, F. Laigret, P. plasmas from flowers. Proceedings of the Republic of China-United Carle, J.M. Bové, R.B. Henegar, N.M. Ellis, D.E. Dodge and J. Adams. States Cooperative Science Seminar, Symposium series 1, National 1998. Entomoplasma freundtii sp. nov., a new species from a green Science Council, Taipei, Taiwan, pp. 75–81. tiger beetle (Coleoptera: Cicindelidae). Int. J. Syst. Bacteriol. 48: McCoy, R.E., H.G. Basham, J.G. Tully, D.L. Rose, P. Carle and J.M. Bové. 1197–1204. 1984. Acholeplasma florum, a new species isolated from plants. Int. J. Weisburg, W.G., J.G. Tully, D.L. Rose, J.P. Petzel, H. Oyaizu, D. Yang, L. Syst. Bacteriol. 34: 11–15. Mandelco, J. Sechrest, T.G. Lawrence, J. Van Etten, J. Maniloff and Pollack, J.D., M.V. Williams, J. Banzon, M.A. Jones, L. Harvey and J.G. C.R. Woese. 1989. A phylogenetic analysis of the mycoplasmas: basis Tully. 1996. Comparative metabolism of Mesoplasma, Entomoplasma, for their classification. J. Bacteriol. 171: 6455–6467. Mycoplasma, and Acholeplasma. Int. J. Syst. Bacteriol. 46: 885–890. Whitcomb, R.F. 1983. Culture media for spiroplasmas. In Methods in Rose, D.L., J.P. Kocka, N.L. Somerson, J.G. Tully, R.F. Whitcomb, P. Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, Carle, J.M. Bové, D.E. Colflesh and D.L. Williamson. 1990. Myco- New York, pp. 147–158. plasma lactucae sp. nov., a sterol-requiring mollicute from a plant sur- Whitcomb, R.F. and K.J. Hackett. 1996. Identification of mollicutes from face. Int. J. Syst. Bacteriol. 40: 138–142. insects. In Molecular and Diagnostic Procedures in Mycoplasmology, Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. measuring growth responses of Mollicutes to serum and polyoxyethyl- 313–322. ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. Whitcomb, R.F., J.G. Tully, D.L. Rose, E.B. Stephens, A. Smith, R.E. Taylor-Robinson, D. 1983. Metabolism inhibition tests. In Methods in McCoy and M.F. Barile. 1982. Wall-less prokaryotes from fall flow- Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, ers in central United States and Maryland. Curr. Microbiol. 7: New York, pp. 411–421. 285–290. Tully, J.G. 1983. Cloning and filtration techniques for mycoplasmas. Williamson, D.L., J.G. Tully, D.L. Rose, K.J. Hackett, R. Henegar, P. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Carle, J.M. Bové, D.E. Colflesh and R.F. Whitcomb. 1990. Mycoplasma Academic Press, New York, pp. 173–177. somnilux sp. nov., Mycoplasma luminosum sp. nov., and Mycoplasma Tully, J.G. 1984. Genus Acholeplasma. In Bergey’s Manual of Systematic lucivorax sp. nov., new sterol-requiring mollicutes from firefly beetles Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, (Coleoptera, Lampyridae). Int. J. Syst. Bacteriol. 40: 160–164. Baltimore, pp. 775–781. 654 Family II. Spiroplasmataceae

Family II. Spiroplasmataceae Skripal 1983, 408VP

Da v i d L. Wi ll i a m s o n , Ga i l E. Ga s p a r i c h , La u r a B. Reg a s s a , Co lle t t e Sa i ll a r d , Jo ë l Re n a u d i n , Jo s e p h M. Bo v é a n d Ro be r t F. Wh i t c o m b * Spi.ro.plas.ma.ta.ce′ae. N.L. neut. n. Spiroplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Spiroplasmataceae the Spiroplasma family. Cells are helical during exponential growth, with rotatory, nicotinamide adenine dinucleotide (NADH) oxidase activity is flexional, and translational motility. Genome size is variable: located only in the cytoplasm. Unable to synthesize fatty acids 780–2220 kbp. Variable sterol requirement for growth. Pro- from acetate. Other characteristics are as described for the cedures for determining sterol requirement are as described class and order. for Family I (Entomoplasmataceae). Possess a phosphoenolpyru- Type genus: Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, vate phosphotransferase system for glucose uptake. Reduced Bové, Tully and Freundt 1973, 201AL.

Genus I. Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 201AL

Da v i d L. Wi ll i a m s o n , Ga i l E. Ga s p a r i c h , La u r a B. Reg a s s a , Co lle t t e Sa i ll a r d , Jo ë l Re n a u d i n , Jo s e p h M. Bo v é a n d Ro be r t F. Wh i t c o m b * Spi.ro.plas¢ma. Gr. n. speira (L. transliteration spira) a coil, spiral; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Spiroplasma spiral form. Cells are pleomorphic, varying in size and shape from helical and branched nonhelical filaments to spherical or ovoid. The helical forms, usually 100–200 nm in diameter and 3–5 mm in length, generally occur during the exponential phase of growth and in some species persist during stationary phase. The cells of some species are short (1–2 mm). In certain cases, helical cells may be very tightly coiled, or the coils may show continu- ous variation in amplitude. Spherical cells ~300 nm in diameter and nonhelical filaments are frequently seen in the stationary phase, where they may not be viable, and in all growth phases in suboptimal growth media, where they may or may not be viable. In some species during certain phases, spherical forms may be the replicating form. Helical filaments are motile, with flexional and twitching movements, and often show an appar- ent rotatory motility. Fibrils are associated with the membrane, but flagellae, periplasmic fibrils, or other organelles of locomo- tion are absent. Fimbriae and pili observed on the cell surface of insect- and plant-pathogenic spiroplasmas are believed to be involved in host-cell attachment and conjugation (Ammar et al., 2004; Özbek et al., 2003), but not in locomotion. Cells FIGURE 111. Colonial morphology of Spiroplasma lampyridicola strain T divide by binary fission, with doubling times of 0.7–37 h. Fac- PUP-l grown on SP-4 agar under anaerobic conditions for 4 d at 30°C. The diffuse appearance and indistinct margins reflect the motility of ultatively anaerobic. The temperature growth range varies spiroplasmas during active growth. Bar = 50 mm. (Reprinted with permis- among species, from 5 to 41°C. Colonies on solid media are sion from Stevens et al., 1997. Int. J. Syst. Bacteriol. 47: 709–712.) frequently diffuse, with irregular shapes and borders, a condi- tion that reflects the motility of the cells during active growth (Figure 111). Colony type is strongly dependent on the agar esculin are not hydrolyzed. Sterol requirements are variable. concentration. Colony sizes vary from 0.1 to 4.0 mm in diam- An optimum osmolality, usually in the range of 300–800 mOsm, eter. Colonies formed by nonmotile variants or mutants, or by has been demonstrated for some spiroplasmas. Media contain- cultures growing on inadequate media are typically umbonate ing mycoplasma broth base, serum, and other supplements are with diameters of 200 mm or less. Some species, such as Spiro- required for primary growth, but after adaptation, growth often plasma platyhelix, have barely visible helicity along most of their occurs in less complex media. Defined or semi-defined media length and display little rotatory or flexing motility. Colonies are available for some species. Resistant to 10,000 U/ml peni- of motile, fast-growing spiroplasmas are diffuse, often with sat- cillin. Insensitive to rifampicin, sensitive to erythromycin and ellite colonies developing from foci adjacent to the initial site tetracycline. Isolated from the surfaces of flowers and other of colony development. Light turbidity may be produced in plant parts, from the guts and hemolymph of various insects liquid cultures. Chemo-organotrophic. Acid is produced from and crustaceans, and from tick triturates. Also isolated from glucose. Hydrolysis of arginine is variable. Urea, arbutin, and vascular plant fluids (phloem sap) and insects that feed on the fluids. Specific host associations are common. The type spe- *Deceased 21 December 2007. cies, Spiroplasma citri, is pathogenic for citrus (e.g., orange and Genus I. Spiroplasma 655 grapefruit), producing “stubborn” disease. Experimental or (Daniels and Longland, 1984, 1980); but motility is random natural infections also occur in horseradish, periwinkle, radish, in the absence of attractants (Daniels and Longland, 1984). broad bean, carrot, and other plant species. Spiroplasma kunkelii Both natural (Townsend et al., 1980b, 1977) and engineered is a maize pathogen. Some species are pathogenic for insects. (Cohen et al., 1989; Duret et al., 1999; Jacob et al., 1997) motil- Certain species are pathogenic, under experimental condi- ity mutants have been described. These mutants form perfectly tions, for a variety of suckling rodents (rats, mice, hamsters and umbonate colonies on solid medium. Mutational analysis has rabbits) and/or chicken embryos. Genome sizes vary from 780 highlighted the involvement of the smc1 gene in motility. Jacob to 2220 kbp (PFGE). et al. (1997) demonstrated that a Tn4001 insertion mutant with

DNA G+C content (mol%): 24–31 (Tm, Bd). reduced flexional motility and no rotational motility could be Type species: Spiroplasma citri Saglio, L’Hospital, Laflèche, complemented with the wild-type scm1 gene. The scm1 gene Dupont, Bové, Tully and Freundt 1973, 202AL. encodes a 409 amino acid polypeptide having ten transmem- brane domains but no significant homology with known pro- Further descriptive information teins. In another study, the scm1 gene was inactivated through Morphology. The morphology of spiroplasmas is most eas- homologous recombination, abolishing motility (Duret et al., − ily observed in suspensions with the light microscope under 1999). The disrupted scm1 mutant was injected into the leaf- dark-field illumination (Williamson and Poulson, 1979). In the hopper vector (Circulifer haematoceps); it multiplied actively in exponential phase in liquid media, most spiroplasma cells are the insect vector and was then transmitted to periwinkle plants. helical filaments 90–250 nm in diameter and of variable length The mutant induced symptoms that were indistinguishable (Figure 112). Fixed and negatively stained cells usually show from those caused by the motile wild-type strain showing that a blunt and a tapered end (Williamson, 1969; Williamson and spiroplasma motility is not essential for phytopathogenicity and Whitcomb, 1974). The tapered ends of the cells are a conse- transmission to the plant host (Duret et al., 1999). quence of the constriction process preceding division (Garnier Fibrils and motility. Microfibrils 3.6 nm in width have been et al., 1981, 1984). However, they are adapted as attachment envisioned in the membranes of some spiroplasmas. These sites in some species (Ammar et al., 2004). structures have repeat intervals of 9 nm along their lengths Motility. Helical spiroplasma cells exhibit flexing, twitching, (Williamson, 1974) and form a ribbon that extends the entire and apparent rotation about the longitudinal axis (Cole et al., length of the helix (Charbonneau and Ghiorse, 1984; William- 1973; Davis and Worley, 1973). Spiroplasmas exhibit tempera- son et al., 1984). The sequence of the fibril protein gene has ture-dependent chemotactic movement toward higher concen- been determined (Williamson et al., 1991) and the calculated trations of nutrients, such as carbohydrates and amino acids mass of the fibril protein is 59 kDa. The flat, monolayered, membrane-bound ribbon composed of several well-ordered fibrils represents the internal spiroplasmal cytoskeleton. The spiroplasmal cytoskeletal ribbon follows the shortest helical line on the cellular coil. Recent studies have focused on the detailed cellular and molecular organization of the cytoskel- eton in Spiroplasma melliferum and Spiroplasma citri (Gilad et al., 2003; Trachtenberg, 2004; Trachtenberg et al., 2003a, b; Tra- chtenberg and Gilad, 2001). Each cytoskeletal ribbon contains seven fibril pairs (or 14 fibrils) and the functional unit is a pair of aligned fibrils (Trachtenberg et al., 2003a). Paired fibrils can be viewed as chains of tetramers composed of 59 kDa mono- mers. Cryo-electron tomography has been used to elucidate the native state, cytoskeletal structure of Spiroplasma melliferum and suggested the presence of three parallel ribbons under the membrane: two appear to be composed of the fibril pro- tein and the third is composed of the actin-like MreB protein (Kürner et al., 2005). Subsequent studies suggest the presence of a single ribbon structure (Trachtenberg et al., 2008). The subunits in the fibrils undergo conformational changes from circular to elliptical, which results in shortening of the fibrils and helix contraction, or from elliptical to circular, leading to a length increase of the fibrils and cell helix. The cytoskeleton, which is bound to the spiroplasmal membrane over its entire length, acts as a scaffold and controls the helical shape of the cell. The cell shape is therefore dynamic. Movement appears to be driven by the propagation of a pair of kinks that travel down the length of the cell along the fibril ribbons (Shaevitz et al., FIGURE 112. Electron micrograph of Drosophila willistoni strain B3SR sex-ratio spiroplasmas. Hemolymph suspension in phosphate buffered 2005; Wada and Netz, 2007; Wolgemuth and Charon, 2005). saline, glutaraldehyde vapor-fixed, and negatively stained with 1% phos- The contractile cytoskeleton can thus be seen as a “linear photungstic acid, pH 7.2. (Reprinted with permission from Whitcomb motor” in contrast to the common “rotary motor” that is part et al., 2007. Biodiversity and Conservation 16: 3877–3894.) of the flagellar apparatus in bacteria (Trachtenberg, 2006). 656 Family II. Spiroplasmataceae

There are several adherent proteins that copurify with the Temperature. ­­ Konai et al. (1996a) determined temperature cytoskeleton, ranging in size from 26 to 170 kDa (Townsend ranges and optima for a large number of spiroplasma strains. et al., 1980a; Trachtenberg, 2006; Trachtenberg and Gilad, The ranges of some strains (e.g., Spiroplasma apis) were very 2001). These proteins are apparently membrane-associated wide (5–41°C), but some group I strains from leafhoppers and and may function as anchor proteins (Trachtenberg and Gilad, plants grew only at 25° and 30°C. Although some spiroplasmas 2001). The structural organization of the cytoskeleton-associ- grew well at 41°C, none grew at 43°C. ated proteins of Spiroplasma melliferum is beginning to be elu- Biochemical reactions. All tested spiroplasmas ferment cidated (Trachtenberg et al., 2008). The 59 kDa polypeptide glucose with concomitant acid production, although the utili- is the cytoskeletal fibril protein. The 26 kDa polypeptide is zation rates may vary. Some strains of group I (e.g., members probably spiralin, the major spiroplasmal membrane protein. of subgroups I-4 and I-6) and all strains of Spiroplasma mirum However, the involvement of spiralin in helicity and motility is ferment glucose slowly. With Spiroplasma citri, all strains tested unlikely (see “Spiralin” section below), especially since spira- grew actively on fructose and strain GII3 grew on fructose, glu- lin is anchored on the outside surface of the cell (Bévén et al., cose, or trehalose. The ability of spiroplasmas to utilize argin- 1996; Bové, 1993; Brenner et al., 1995; Foissac et al., 1996) and ine varies (Hackett et al., 1996a). Arginine hydrolysis by some spiralin-deficient mutants maintain helicity and motility (Duret spiroplasmas can be observed only if glucose is also present et al., 2003). The 45 kDa protein may correspond to the prod- in the medium. In other cases, aggressive glucose metabolism uct of the scm1 gene, shown to be essential for motility (Jacob interferes with detection of arginine hydrolysis (Hackett et al., et al., 1997), and the 34 kDa protein may be the product of the 1996a). mreB1 gene (W. Maccheroni and J. Renaudin, unpublished). MreB is the bacterial homolog to eukaryotic actin (Jones et al., Regulation of the fructose and trehalose operons of Spiroplasma 2001; Van den Ent et al., 2001). Early work provided evidence citri. The fructose operon of Spiroplasma citri (Gaurivaud for the presence of actin-like proteins in spiroplasmas. Antisera et al., 2000a) became of special interest when fructose utiliza- prepared against SDS-denatured invertebrate actin coupled to tion was implicated in Spiroplasma citri phytopathogenicity (see horseradish peroxidase specifically stained cells of Spiroplasma citri “Mechanism of Spiroplasma citri phytopathogenicity” below). (Williamson et al., 1979a). Also, a protein with a molecular mass In particular, the role of the first gene of the operon, fruR, similar to that of actin (protein P25) was isolated from Spiroplasma was investigated. In vivo transcription of the operon is greatly citri and reacted with IgG directed against rabbit actin (Mouches enhanced by the presence of fructose in the growth medium, et al., 1982b, c, 1983b). Monospecific antibodies raised against whereas glucose has no effect. When fruR is not expressed the P25 protein recognized not only P25 of Spiroplasma citri, but (fruR− mutants), transcription of the operon is not stimulated also a homologous protein from Mycoplasma mycoides PG50 and by fructose and the rate of fructose fermentation is decreased, Ureaplasma urealyticum serotype V (Mouches et al., 1983b). More indicating that FruR is an activator of the fructose operon (Gau- recent work has focused on the molecular organization of the rivaud et al., 2001). Trehalose is the major sugar in leafhoppers genes. mreB genes are present in rod-shaped, filamentous, and and other insects. The trehalose operon of Spiroplasma citri has helical bacteria, but not in coccoid, spherical bacteria, regard- a gene organization very similar to that of the fructose operon less of whether or not they are Gram-stain-positive or Gram- and the first gene of the trehalose operon, treR, also encodes a stain-negative. mreB genes are also absent from the pleomorphic transcriptional activator of the operon (André et al., 2003). mycoplasmas. However, Spiroplasma citri contains five homologs Sterol utilization. It was originally thought that all spiroplas- of Bacillus subtilis mreB genes (Maccheroni et al., 2002). Four of mas require sterol for growth. Subsequent screening by Rose these (mreB2, 3, 4, and 5) form a cluster on the genome and are et al. (1993) showed that a minority of the spiroplasmas tested transcribed in two separate operons. Gene mreB1 is transcribed as were able to sustain growth in mycoplasma broth base medium a monocistronic operon and at a much higher level. without sterols. The discovery that the sterol requirement in Growth characteristics. Spiroplasma cells increase in length Mollicutes is polyphyletic greatly diminished the significance of and divide by constriction. Pulse labeling of the membrane with sterol requirements in mollicute taxonomy (Tully et al., 1993). tritiated amino acids revealed a polar growth of the helix. Polar- Metabolic pathways and enzymes. The intermediary meta­ ity was also observed by tellurium-labeling of oxido-reduction bolism of Mollicutes has been reviewed (Miles, 1992; Pollack, sites (Garnier et al., 1984). In the stationary or death phase, the 2002a, b; Pollack et al., 1997). Like all mollicutes, Spiroplasma cells are usually distorted, often forming either subovoid bod- species apparently lack both cytochromes and, except for malate ies or nonhelical filaments. Within cultured insect cells, all the dehydrogenase, the enzymes of the tricarboxylic acid cycle. spiroplasma cells were subovoid, but presumably viable (Waya- They do not have an electron-transport system and their res- dande and Fletcher, 1998). Thus, the ability of cells to grow and piration is characterized as being flavin-terminated. McElwain divide is not linked inextricably to helicity. et al. (1988) studied Spiroplasma citri and Pollack et al. (1989) Growth rate. Enumerated microscopically (Rodwell and screened ten spiroplasma species for 67 enzyme activities. All Whitcomb, 1983), spiroplasmas reach titers of 108–1011 cells/ spiroplasmas were fermentative; their 6-phosphofructokinases ml in medium containing horse or fetal bovine serum. Growth (6-PFKs) required ATP for substrate phosphorylation during rates of related strains tend to be similar. Konai et al. (1996a) glycolysis. This enzymic requirement is common to all molli- calculated doubling times from the time required for medium cutes except Acholeplasma and Anaeroplasma spp. The 6-PFKs of acidification. In general, spiroplasmas adapted to complex the species in these genera require pyrophosphate and cannot cycles or single hosts had slower growth rates than spiroplasmas use ATP. Additionally, except for Spiroplasma floricola, all Spiro- known or suspected to be transmitted on plant surfaces. plasma species have dUTPase activity. Pollack et al. (1989) also Genus I. Spiroplasma 657 reported that all spiroplasmas except Spiroplasma floricola have nucleotides are not uniformly distributed over the genome (Ye deoxyguanosine kinase activity. They found that deoxyguanos- et al., 1992). ine, but no other nucleoside, could be phosphorylated to GMP Methylated bases. Methylated bases have been detected in with ATP. spiroplasmal DNA (Nur et al., 1985). The gene encoding the Spiroplasmal proteins with multiple functions have been CpG methylase in Spiroplasma monobiae has been cloned (Ren- described. The CpG-specific methylase from Spiroplasma mono- baum et al., 1990) and its mode of action studied (Renbaum biae appears to also have topoisomerase activity (Matsuo et al., and Razin, 1992). 1994). Protein P46 of Spiroplasma citri is a bifunctional protein in which the N-terminal domain represents ribosomal pro- DNA restriction patterns. Restriction patterns of spiroplas- tein L29, whereas the C-terminal domain is capable of bind- mal DNA, as determined by polyacrylamide gel electrophoresis, ing a specific inverted repeat sequence. It could be involved may be highly similar among strains of a given species (Bové in regulation (Le Dantec et al., 1998). Such protein multifunc- et al., 1989). Variations in restriction fragment length patterns tionality may reflect genomic economy in the small mollicute among strains of Spiroplasma corruscae correlated imperfectly genome (Pollack, 2002b). However, functional redundancy has with serological variation, so their significance was uncertain also been reported; Spiroplasma citri apparently has two distinct (Gasparich et al., 1998). membrane ATPases (Simoneau and Labarère, 1991). RNA genes. Some spiroplasmas, such as Spiroplasma citri, Genome size, genomic maps, and chromosomal rearrange- have only one rRNA operon, whereas others, such as Spiro- ments. PFGE revealed that the genome size range for spiro- plasma apis, have two (Amikam et al., 1984, 1982; Bové, 1993; plasmas varied continuously (Pyle and Finch, 1988) from 780 Grau et al., 1988; Razin, 1985). The three rRNA genes are kbp for Spiroplasma platyhelix to 2220 kbp for Spiroplasma ixodetis linked in the classical order found in bacteria: 5¢-16S–23S-5S-3¢. (Carle et al., 1995, 1990). There is a general trend for genomic The sequence of the 16S rRNA gene (rDNA) of most spiro- simplification in Spiroplasma lineages. This trend culminated in plasma species has been determined for phylogenetic studies loss of helicity and motility in the Entomoplasmataceae and even- (Gasparich et al., 2004; Weisburg et al., 1989). A gene cluster of tually to the host transfer events forming the mycoides group of ten tRNAs (Cys, Arg, Pro, Ala, Met, Ile, Ser, fMet, Asp, Phe) was mycoplasmas (Gasparich et al., 2004). identified in Spiroplasma melliferum (Rogers et al., 1987). Simi- The genome size of Spiroplasma citri varies among strains lar tRNA gene clusters have been cloned and sequenced from from 1650 to 1910 kbp (Ye et al., 1995). It was found that the Spiroplasma citri (Citti et al., 1992). relative positions of mapped loci were conserved in most of the Codon usage. In spiroplasmas, UGA is not a stop codon but strains, but that differences in the sizes of certain fragments encodes tryptophan. The universal tryptophan codon, UGG, is permitted genome size variation. Genome size can fluctuate also used (Citti et al., 1992; Renaudin et al., 1986). Codon usage rapidly in spiroplasma cultures after a relatively short number also reflects the A+T richness of spiroplasmal DNA (usually of in vitro passages (Melcher and Fletcher, 1999; Ye et al., 1996). about 74 mol% A+T). For example, in Spiroplasma citri, UGA is The genome of Spiroplasma melliferum is 360 kbp shorter than used to code for tryptophan eight times more frequently than that of Spiroplasma citri strain R8-A2T, but DNA hybridization has the universal tryptophan codon UGG (Bové, 1993; Citti et al., shown that the two spiroplasmas share extensive DNA hybridiza- 1992; Navas-Castillo et al., 1992). Also, synonymous codons with tion (65%). Comparison of their genomic maps revealed that U or A at the 5¢ or 3¢ ends are preferentially used over those the genome region, which is shorter in Spiroplasma melliferum, with a C or G in that position. corresponds to a variable region in the genomes of Spiroplasma RNA polymerase and spiroplasmal insensitivity to rifam- citri strains and that a large region of the Spiroplasma melliferum picin. Spiroplasmas are insensitive to rifampicin. DNA- genome is inverted in comparison with Spiroplasma citri. There- dependent RNA polymerases from Spiroplasma melliferum and fore, chromosomal rearrangements and deletions were proba- Spiroplasma apis were at least 1000 times less sensitive to rifam- bly major events during evolution of the genomes of Spiroplasma picin than the corresponding Escherichia coli enzyme (Gadeau citri and Spiroplasma melliferum. In addition, a large amount of et al., 1986). Rifampicin insensitivity of Spiroplasma citri and noncoding DNA is present as repeat sequences (McIntosh et al., all other mollicutes tested was found to be associated with the 1992; Nur et al., 1986, 1987) and integrated viral DNA (Bébéar presence of an asparagine residue at position 526 in RpoB. et al., 1996) may also account for differences in genome sizes of The importance of the asparagine residue was confirmed by closely related species. site-directed mutagenesis of the histidine codon (CAC) to Base composition. The DNA G+C content for most spiro- an asparagine codon (AAC) at position 526 of Escherichia coli plasma groups and subgroups has been determined (Carle RpoB, resulting in a rifampicin-resistant mutant (Gaurivaud et al., 1995, 1990; Williamson et al., 1998). Most group I spiro- et al., 1996). The genetic organization surrounding the rpoB plasmas and Spiroplasma poulsonii have a G+C content of 25–27 gene in spiroplasmas is also atypical. In many bacteria, rpoB is mol%. However, the G+C content of subgroup I-6 Spiroplasma part of the b operon in which the four genes rplK, rplA, rplJ, insolitum is significantly higher, indicating that the base compo- and rplL, encoding ribosomal proteins L11, L1, L10, and L12, sition of spiroplasmal DNA may shift over relatively short evo- respectively, are located immediately upstream of rpoB; rpoC is lutionary periods. The range of G+C content of 25–27 mol% immediately downstream of rpoB. In Spiroplasma citri, the gene is modal for Spiroplasma and is also common in the Apis clade. organization is different in that the hsdS gene, encoding a com- However, Spiroplasma mirum (group V), strains of Spiroplasma ponent of a type I restriction-modification system, is upstream apis (group IV), and group VIII strains have a G+C content of of rpoB. Sequences showing similarities with insertion elements about 29–31 mol%. Restriction sites containing only G and C are found between hsdS and rpoB (Laigret et al., 1996). 658 Family II. Spiroplasmataceae

DNA polymerases and other proteins involved in DNA rep- region and an amino acid sequence repetition, including a lication and repair. From genomic studies, it appears that VTKXE consensus sequence, are present in all spiralins analyzed Mycoplasma species carry the essential, multimeric enzyme for (Foissac et al., 1997a). Spiralin confers a significant amount of genomic DNA replication, DNA polymerase III. The subunit the antigenic activity in group I spiroplasmas (Whitcomb et al., responsible for actual DNA biosynthesis is subunit a, encoded 1983) and has a high degree of species specificity, although by polC (dnaE). The polC gene has been identified in all minor cross-reactions have been detected (Zaaria et al., 1990). sequenced mollicute genomes, including Spiroplasma citri. The The spiralin genes of Spiroplasma citri and Spiroplasma melliferum genes encoding the other subunits, dnaN (subunit b) and dnaX species, which have about 65% overall DNA–DNA hybridization, (subunits t and g), are also shared by the Spiroplasma and Myco- shared 89% nucleotide sequence identity and 75% deduced plasma species studied to date. So, it seems that spiroplasmas, amino acid sequence similarity (Bové et al., 1993). like other mollicutes, possess DNA polymerase III and that it is Spiralin mutants were constructed through homologous probably the major DNA replication enzyme. However, there recombination in Spiroplasma citri to examine the role of spi- is also evidence for two additional DNA polymerases. A second ralin in vivo (Duret et al., 2003). Phenotypic characterization gene for a DNA polymerase (enzyme B) was found in the Spiro- of mutant 9a2 showed that, in spite of a total lack of spiralin, it plasma citri genome and there is evidence that the Spiroplasma maintained helicity and motility similar to the wild-type strain kunkelii polA gene may encode a full-length DNA polymerase GII3 (Duret et al., 2003). When injected into the leafhopper I protein (Bai and Hogenhout, 2002). DNA polymerase I is a vector, Circulifer haematoceps, the mutant multiplied to a high single polypeptide that has, in addition to DNA synthesis activ- titer, but transmission efficiency to periwinkle plants was very ity, two exonuclease activities: exo-3¢ to 5¢ as well as exo-5¢ to low compared to the wild-type strain. In the infected plants, 3¢. At this stage, it is not possible to determine the equivalence however, the spiralin-deficient mutant multiplied well and pro- between the three spiroplasmal DNA polymerases identified by duced the typical symptoms of the disease. In addition, prelimi- sequencing (Pol III, enzyme B, and Pol I) and those originally nary results indicated that the mutant could not be acquired detected biochemically (ScA, ScB, ScC) (Charron et al., 1979, by insects feeding on 9a2-infected plants, suggesting that spira- 1982). As the Spiroplasma citri genome sequencing project has lin may mediate spiroplasma invasion of insect tissues (Duret progressed, the following Spiroplasma citri genes involved in et al., 2003). In order to test this possibility, Circulifer haemato- DNA replication have been detected: dnaA, dnaB, polA, dnaE, ceps leafhopper proteins were screened as putative Spiroplasma polC, dnaN, dnaX, holB, dinB (truncated), dnaJ, dnaK, gyrA, gyrB, citri-binding molecules using Far-Western analysis (Killiny et al., parC, parE, topA, rnhB, rnhC, rnpA, rnR, rnc, yrrc, xseA, xseB, and ssb 2005).These experiments showed that spiralin is a lectin capa- (Carle et al., 2010; accession numbers AM285301–AM285339). ble of binding to insect 50 and 60 kDa mannose glycoproteins. Genes encoding DNA replication proteins have also been iden- Hence, spiralin could play a key role in insect transmission of tified in Spiroplasma kunkelii (Bai and Hogenhout, 2002). Spiro- Spiroplasma citri by mediating spiroplasma adherence to epithe- plasma citri is highly sensitive to UV irradiation (Labarère and lial cells of the insect vector gut or salivary gland (Killiny et al., Barroso, 1989) and the organism has no functional recA gene, 2005). This would also explain why the spiralin-negative mutant since a significant portion of the C-terminal part of the gene is 9a2 is poorly transmitted by the vector and is not acquired by lacking (Marais et al., 1996). insects feeding on 9a2-infected plants.

Origin of DNA replication. Even before the Spiroplasma Viruses. Four different virus types have been found in Spiro- citri genome project was initiated, some fragments with mul- plasma, SpV1-SpV4. Use of SpV1 viruses for recombinant DNA tiple open reading frames had been completely sequenced. studies in Spiroplasma citri is described later in the section on For example, Ye et al. (1994b) sequenced a 5.6 kbp fragment “Tools for molecular genetics of Spiroplasma citri ”. containing genes for the replication initiation protein (dnaA), Cells of many spiroplasma species contain filamentous/rod- the beta subunit of DNA polymerase III (dnaN), and the DNA shaped viruses (SVC1 = SpV1) that are associated with nonlytic gyrase subunits A and B (gyrA and gyrB). Several dnaA-box con- infections (Bové et al., 1989; Ranhand et al., 1980; Renaudin sensus sequences were found upstream and downstream of the and Bové, 1994). They belong to the Plectrovirus group within dnaA gene. From these data, it was established that the dnaA the Inoviridae. SpV1 viruses have circular, single-stranded DNA region was the origin of replication in Spiroplasma citri (Ye et al., genomes (7.5 to 8.5 kbp), some of which have been sequenced 1994b). Zhao et al. (2004a) cloned a cell division gene cluster (Renaudin and Bové, 1994). SpV1 sequences also occur as from Spiroplasma kunkelii and functionally characterized the key prophages in the genome of the majority of Spiroplasma citri strains studied (Renaudin and Bové, 1994). These insertions division gene, ftsZsk, and showed that it encodes a cell division protein similar to FtsZ proteins from other bacteria. take place at numerous sites in the chromosomes of Spiroplasma citri (Ye et al., 1992) and Spiroplasma melliferum (Ye et al., 1994a). Spiralin. Spiralin, encoded by the spi gene, is the major The SpV1-ORF3 and the repeat sequences could be part of an membrane protein of Spiroplasma citri (Wróblewski et al., 1977, IS-like element of chromosomal origin. Resistance of spiroplas- 1989). The deduced amino acid sequence of the protein (Bové mas to virus infection may be associated with integration of et al., 1993; Chevalier et al., 1990; Saillard et al., 1990) corre- viral DNA sequences in the chromosome or extrachromosomal sponds well with the experimentally determined amino acid elements (Sha et al., 1995). The evolutionary history of these composition (Wróblewski et al., 1984). In particular, spiralin viruses is unclear, but there is some evidence for virus and plas- lacks tryptophan and, thus, has no UGG and/or UGA codons, mid co-evolution in the group I Spiroplasma species (Gasparich which facilitates gene expression in Escherichia coli. Detailed et al., 1993a) and indications of potentially widespread horizon- analyses showed that all Spiroplasma citri spiralins were 241–242 tal transmission (Vaughn and de Vos, 1995). Virus infection of amino acids long (Foissac et al., 1996). A conserved central spiroplasma cells can pose problems in cultures. For example, Genus I. Spiroplasma 659 lyophilized early passages of Spiroplasma citri R8-A2T proved plasmids: pSciA (7.8 kbp), pSci1 to pSci6 (12.9 to 35.3 kbp), difficult to grow and electron microscopy revealed that these and one viral RF DNA (SVTS2). The chromosomal contigs con- cells carried large numbers of virions of virus SpV1-R8A2 (Cole tained 1905 putative genes or coding sequences (CDS). Of the et al., 1974). Likewise, SpV1 viruses have been found in Spiro- CDS-encoded proteins, 29% are involved in cellular processes, plasma poulsonii (Cohen et al., 1987) and Spiroplasma melliferum cell metabolism, or cell structure. CDS for viral proteins and (Liss and Cole., 1981); the Spiroplasma melliferum SpV1-KC3 mobile elements represented 24% of the total, whereas 47% of virus forms plaques on various strains of Spiroplasma melliferum, the CDS were for hypothetical proteins with no known function; including the type strain BC-3T. 21% of the total CDS appeared truncated as compared to their A second virus, reminiscent of a type B tailed bacterial virus, bacterial orthologs. Families of paralogs were mainly clustered occurs in a small number of Spiroplasma citri strains (Cole et al., in a large region of the chromosome opposite the origin of rep- 1973). This SCV2 (= SpV2) virus is a polyhedron with a long, lication. Eighty-four CDS were assigned to transport functions, noncontractile tail. It may be associated with lytic infection. including phosphoenolpyruvate phosphotransferase systems Infections in which large numbers of virions of SpV2 viruses are (PTS), ATP binding cassette (ABC) transporters, and ferritin. In produced tend to be irregular and difficult to maintain under addition to the general enzymes EI and HPr, glucose- fructose- experimental conditions, so this is the least studied of the spiro- and trehalose-specific PTS permeases, and glycolytic and ATP plasma viruses. synthesis pathways, Spiroplasma citri possesses a Sec-dependent A third virus (SpV3) forms polyhedral virions with short protein export system and a nearly complete pathway for ter- tails and has been found in many strains of Spiroplasma citri penoid biosynthesis. The sequencing of the Spiroplasma kunkelii (Cole, 1979, 1977, 1974). The SpV3 genome is a linear dou- CR2-3x genome (1.55 Mb) is also nearing completion (http:// ble-stranded DNA molecule of 16 kbp, which can circularize www.genome.ou.edu/spiro.html); the physical and genetic to form a covalently closed molecule with single-stranded gaps, maps have been published (Dally et al., 2006). Several studies indicating that the linear molecule has cohesive ends. There have begun to focus on gene content and genomic organiza- is significant diversity among SpV3 viruses, extending even to tion (Zhao et al., 2003, 2004a, b). Results show that, in addition major differences in genome sizes. Virus SpV3-AV9/3 was iso- to virus SpV1 DNA insertions, the Spiroplasma kunkelii genome lated from Spiroplasma citri strain ASP-9 (Stephens, 1980). Dick- harbors more purine and amino acid biosynthesis, transcrip- inson and Townsend (1984) isolated the SpV3 virus from plants tional regulation, cell envelope, and DNA transport/binding infected with Spiroplasma citri. This virus, when plated on cells of genes than Mycoplasmataceae (e.g., Mycoplasma genitalium and Spiroplasma citri, had a plaque morphology typical of temperate Mycoplasma pneumoniae) genomes (Bai and Hogenhout, 2002). phages. In spiroplasma cells that have been lysogenized, com- Plasmids. Several plasmids have been discovered in spiro- plete virus genomes may be integrated into the spiroplasma plasmas (Archer et al., 1981; Gasparich and Hackett, 1994; chromosome. These cells are then immune to superinfection Gasparich et al., 1993a; Mouches et al., 1984a; Ranhand et al., by the lysogenizing virus, but susceptible to other SpV3 viruses. 1980). They are especially common in spiroplasmas of group It is possible that lysogenization of Spiroplasma citri by SpV3-ai I. Eight extrachromosomal elements, including seven plas- affects spiroplasma pathogenicity, particularly with respect to mids, were discovered during the Spiroplasma citri GII3 genome attenuation. Drosophila spiroplasmas, male-lethal or nonlethal, sequencing project. The six largest plasmids, pSci1 to pSci6, usually carry SpV3 viruses. Each strain of Drosophila spiroplasma range from 12.9 to 35.3 kb (Saillard et al., 2008). In silico carries an associated virus that is lytic to certain other strains analyses of plasmid sequences revealed that they share exten- (Oishi et al., 1984). sive regions of homology and display a mosaic gene organiza- A fourth virus (SpV4), with a naked, icosahedral nucleo- tion. Genes encoding proteins of the TraD-TraG, TrsE-TraE, capsid 25 nm in diameter, was discovered (Ricard et al., 1982) and Soj-ParA protein families, were predicted in most of the in the B63 strain of Spiroplasma melliferum. SpV4 has a circu- pSci sequences. The presence of such genes, usually involved lar, single-stranded DNA genome (Renaudin and Bové, 1994; in chromosome integration, cell to cell DNA transfer, or DNA Renaudin et al., 1984a, b) and is a lytic Spiromicrovirus within element partitioning; suggests that these molecules could be the Microviridae (Chipman et al., 1998). Infection with this virus inherited vertically as well as horizontally. The largest plasmid, results in very clear plaques, indicating a lytic process. Host pSci6, encodes P32 (Killiny et al., 2006), a membrane-associ- range studies (Renaudin et al., 1984a, b) have shown that only ated protein interestingly absent in all insect non-transmissible Spiroplasma melliferum is susceptible to SpV4. Two strains of Spiro- strains tested so far. The five remaining plasmids (pSci1 to T plasma melliferum, including the type strain BC-3 and B63, are pSci5) encode eight different Spiroplasma citri adhesion-related not susceptible, as no plaques were formed on lawns of these proteins. The complete sequences of plasmids pSKU146 from spiroplasmas. These strains could be infected by transfection Spiroplasma kunkelii CR2-3x and pBJS-O from Spiroplasma citri suggesting that resistance to the whole virus occurred at the BR3 have been reported (Davis et al., 2005; Joshi et al., 2005). level of adsorption or penetration of the virus (Renaudin and These large plasmids, like the above Spiroplasma citri plasmids, Bové, 1994; Renaudin et al., 1984b). encode an adhesin and components of a type IV translocation- Genome sequencing. Genomic DNA sequencing efforts related conjugation system. Characterizing the replication and for two Spiroplasma species are in progress. For Spiroplasma citri stability regions of Spiroplasma citri plasmids resulted in the iden- GII3 (Carle et al., 2010; Saillard et al., 2008), assembly of 20,000 tification of a novel replication protein, suggesting that Spiro- sequencing reads obtained from shotgun and chromosome plasma citri plasmids belong to a new plasmid family and that specific libraries yielded: (1) 39 chromosomal contigs totalling the soj gene is involved in segregational stability of these plas- 1525 kbp of the 1820 kbp Spiroplasma citri GII3 chromosome as mids (Breton et al., 2008a). Similar replicons were detected in well as (2) 8 circular contigs, which proved to represent seven various spiroplasmas of group I, such as Spiroplasma ­melliferum, 660 Family II. Spiroplasmataceae

Spiroplasma kunkelii, Spiroplasma sp. 277F, and Spiroplasma phoeni- IICB component of the glucose phosphotransferase system ceum, showing that they are not restricted to plant pathogenic permease (ptsG) (André et al., 2005; Duret et al., 2003; Lar- spiroplasmas. tigue et al., 2002). Another series of recombinant plasmids, the Tools for molecular genetics of Spiroplasma citri. Recent pGOT vectors, allow for selection of rare recombination events recombinant DNA tools are described in this section. by using two distinct selective markers. First, transformants are Several reports have been published concerning the use screened for their resistance to gentamicin and next, site-spe- of SpV1 viruses as tools to introduce recombinant DNA into cific recombinants are selected for based on their resistance to spiroplasmas, including optimization of transfection conditions tetracycline, which can only be expressed through recombina- (Gasparich et al., 1993b). The replicative form of SpV1 was tion at the target gene. In this way, inactivation of the crr gene, used to clone and express the Escherichia coli-derived chloram- encoding the glucose phosphotransferase permease IIA compo- phenicol acetyltransferase (cat) gene in Spiroplasma citri. Both nent, was obtained (Duret et al., 2005). The use of the transpo- gd the replicative form (RF) and the virion DNA produced by the son TnpR/res recombination system to produce unmarked transfected cells contained the cat gene sequences (Stambur- mutations (i.e., without insertion of antibiotic markers) in Spiro- ski et al., 1991). The G fragment of the Mycoplasma pneumoniae plasma citri was demonstrated by the production of a disrupted cytadhesin P1 gene could also be expressed in Spiroplasma citri arcA mutant (Duret et al., 2005); arcA encodes arginine deimi- (Marais et al., 1993) using a similar method. However, the nase. In this system, the target gene is disrupted by integration recombinant RF proved unstable, resulting in the loss of the of a plasmid containing target gene sequences along with the DNA insert (Marais et al., 1996). tetM gene flanked by binding-specific recombination (res) sites. Recombinant plasmids have also been developed to intro- After integration of the plasmid, a second plasmid is introduced duce genes into Spiroplasma citri cells. The introduced genes that encodes the resolvase TnpR. TnpR mediates the resolution include antibiotic resistance markers and wild-type genes to of the cointegrate at the res sites, thereby removing tetM but complement auxotrophic mutants. Most recombinant plas- leaving behind a mutated version of the target gene. The TnpR- mids contain the origin of DNA replication (oriC) of the Spiro- encoding plasmid is lost spontaneously when selective pressure plasma citri chromosome (Ye et al., 1994b). One such plasmid is removed. is pBOT1 (Renaudin, 2002; Renaudin et al., 1995). This plas- Antigenic structure. Growth inhibition tests (Whitcomb mid contains a 2 kbp oriC region, a tetracycline resistance gene et al., 1982) were used in the early years to identify spiroplasma (tetM) from Tn916, and the linearized Escherichia coli plasmid species or groups, but metabolism inhibition (Williamson et al., pBS with a colE1 origin of replication. Because of its two ori- 1979b; Williamson and Whitcomb, 1983) and deformation tests gins of replication, oriC and colE1, pBOT1 is able to shuttle (Williamson et al., 1978) are now used almost exclusively (see between Spiroplasma citri and Escherichia coli. When introduced below). into Spiroplasma citri, pBOT1 replicates first as a free extrachro- Antigenic variability, which has been described for some mosomal element, but later integrates into the chromosome via Mycoplasma species (Rosengarten and Wise, 1990; Yogev et al., homologous recombination involving a single crossover event 1991), has not been demonstrated in spiroplasmas (R. Rosen- in the oriC region. Once integrated into the host chromosome, garten, personal communication). the whole plasmid is stably maintained. Recent studies suggest that the broad host range Spiroplasma citri GII3 plasmids and Group classification. The classification of spiroplasmas was their shuttle derivatives may have significant advantages over first proposed by Junca et al. (1980) and has been revised peri- oriC plasmids for gene transfer and expression in spiroplasmas odically (Tully et al., 1987; Williamson et al., 1998). These clas- (Breton et al., 2008a). They transform Spiroplasma citri (as well sifications are based on serological reactions of the organisms as Spiroplasma kunkelii and Spiroplasma phoeniceum) strains at in growth inhibition, deformation and metabolism inhibition relatively high efficiencies, the growth of the transformants is tests and/or characteristics of their genomes. Development not significantly affected, they do not integrate into the chro- of a classification scheme has resulted in the delineation of mosome, and their stability/loss can be modulated depending spiroplasma groups and subgroups (Table 142). In the scheme, upon the presence/absence of the soj gene. “groups” have been defined as clusters of similar organisms, Spiroplasma citri mutants have been produced by random and all of which possess negligible DNA–DNA hybridization with targeted approaches. The transposon Tn4001 has been used representatives of other groups, but moderate to high levels of successfully for random mutagenesis of Spiroplasma citri (Foissac hybridization (20–100%) with each other. Groups are, there- et al., 1997c). For targeted gene inactivation, plasmids derived fore, putative species. This level of genomic differentiation cor- from pBOT1 have been used to disrupt genes (e.g., fructose relates well with substantial differences in serology. Thirty-four operon, motility gene scm1) through homologous recombi- groups were presented in a revised classification of spiroplas- nation involving a single crossover event (Duret et al., 1999; mas in 1998 (Williamson et al., 1998). Four additional groups Gaurivaud et al., 2000c). More recently, Lartigue et al. (2002) (XXXV–XXXVIII) were proposed recently as the result of a developed vector pC2, in which the oriC fragment was reduced global spiroplasma environmental survey (Whitcomb et al., to the minimal sequence needed to promote plasmid replica- 2007) and more are anticipated (Jandhyam et al., 2008). Sub- tion; this vector increases recombination frequency at the tar- groups have been defined by the International Committee on get gene. To avoid the extensive passaging that was required for Systematics of Bacteria (ICSB) Subcommittee on the Taxonomy recombination prior to transformant screening, vector pC55 of Mollicutes (ICSB, 1984) as clusters of spiroplasma strains was designed using a selective tetracycline resistance marker showing intermediate levels of intragroup DNA–DNA hybrid- that is only expressed after the plasmid has integrated into the ization (10–70%) and possessing corollary serological relation- chromosome at the target gene. This approach was used to ships. Three spiroplasma groups [group I (Junca et al., 1980; inactivate the spiralin gene (spi) and the gene encoding the Saillard et al., 1987), group VIII (Gasparich et al., 1993c), and Genus I. Spiroplasma 661

Table 142. Biological properties of spiroplasmasa

Group Spiroplasma Strain ATCC no. Morphologyb Genomec G+Cd Arge Dtf OptTg Host I-1 S. citri R8-A2T 27556T Long helix 1820 26 + 4.1 32 Phloem/leafhopper I-2 S. melliferum BC-3T 33219T Long helix 1460 26 + 1.5 37 Honey bee I-3 S. kunkelii E275T 29320T Long helix 1610 26 + 27.3 30 Phloem/leafhopper I-4 Spiroplasma sp. 277F 29761 Long helix 1620 26 + 2.3 32 Rabbit tick I-5 Spiroplasma sp. LB-12 33649 Long helix 1020 26 − 26.3 30 Plant bug I-6 S. insolitum M55T 33502T Long helix 1810 28 − 7.2 30 Flower surface I-7 Spiroplasma sp. N525 33287 Long helix 1780 26 + 4.7 32 Green June beetle I-8 S. phoeniceum P40T 43115T Long helix 1860 26 + 16.8 30 Phloem/vector I-9 S. penaei SHRIMPT BAA-1082T Helix nd 29 + nd 28 Pacific white shrimp (CAIM 1252T) II S. poulsonii DW-1T 43153T Long helix 1040 26 nd 15.8 30 Drosophila hemolymph III S. floricola OMBG 29989T Helix 1270 26 − 0.9 37 Plant surface IV S. apis B31T 33834T Helix 1300 30 + 1.1 34.5 Honey bee V S. mirum SMCAT 29335T Helix 1300 30 + 7.8 37 Rabbit tick VI S. ixodetis Y32T 33835T Tight coil 2220 25 − 9.2 30 Ixodid tick VII S. monobiae MQ-1T 33825T Helix 940 28 − 1.9 32 Monobia wasp VIII-1 S. syrphidicola EA-1T 33826T Minute helix 1230 30 + 1.0 32 Syrphid fly VIII-3 Spiroplasma sp. TAAS-1 51123 Minute helix 1170 31 + 1.4 37 Horse fly VIII-2 S. chrysopicola DF-1T 43209T Minute helix 1270 29 + 6.4 30 Deer fly IX S. clarkii CN-5T 33827T Helix 1720 29 + 4.3 30 Green June beetle X S. culicicola AES-1T 35112T Short helix 1350 26 − 1.0 37 Mosquito XI S. velocicrescens MQ-4T 35262T Short helix 1480 26 − 0.6 37 Monobia wasp XII S. diabroticae DU-1T 43210T Helix 1350 25 + 0.9 32 Beetle XIII S. sabaudiense Ar-1343T 43303T Helix 1175 29 + 4.1 30 Mosquito XIV S. corruscae EC-1T 43212T Helix nd 26 − 1.5 32 Horse fly/beetle XV Spiroplasma sp. I-25 43262 Wave-coil 1380 26 − 3.4 30 Leafhopper XVI-1 S. cantharicola CC-1T 43207T Helix nd 26 − 2.6 32 Cantharid beetle XVI-2 Spiroplasma sp. CB-1 43208 Helix 1320 26 − 2.6 32 Cantharid beetle XVI-3 Spiroplasma sp. Ar-1357 51126 Helix nd 26 − 3.4 30 Mosquito XVII S. turonicum Tab4cT 700271T Helix 1305 25 − nd 30 Horse fly XVIII S. litorale TN-1T 43211T Helix 1370 25 − 1.7 32 Horse fly XIX S. lampyridicola PUP-1T 43206T Unstable helix 1375 25 − 9.8 30 Firefly XX S. leptinotarsae LD-1T 43213T Motile funnel 1085 25 + 7.2 30 Colorado potato beetle XXI Spiroplasma sp. W115 43260 Helix 980 24 − 4.0 30 Flower surface XXII S. taiwanense CT-1T 43302T Helix 1195 26 − 4.8 30 Mosquito XXIII S. gladiatoris TG-1T 43525T Helix nd 26 − 4.1 31 Horse fly XXIV S. chinense CCHT 43960T Helix 1530 29 − 0.8 37 Flower surface XXV S. diminutum CUAS-1T 49235T Short helix 1080 26 − 1.0 32 Mosquito XXVI S. alleghenense PLHS-1T 51752T Helix 1465 31 + 6.4 30 Scorpion fly XXVII S. lineolae TALS-2T 51749T Helix 1390 25 − 5.6 30 Horse fly XXVIII S. platyhelix PALS-1T 51748T Wave-coil 780 29 + 6.4 30 Dragonfly XXIX Spiroplasma sp. TIUS-1 51751 Rare helices 840 28 − 3.6 30 Tiphiid wasp XXX Spiroplasma sp. BIUS-1 51750 Late helices nd 28 − 0.9 37 Flower surface XXXI S. montanense HYOS-1T 51745T Helix 1225 28 + 0.7 32 Horse fly XXXII S. helicoides TABS-2T 51746T Helix nd 27 − 3.0 32 Horse fly XXXIII S. tabanidicola TAUS-1T 51747T Helix 1375 26 − 3.7 30 Horse fly XXXIV Spiroplasma sp. B1901 700283 Helix 1295 25 − nd nd Horse fly XXXV Spiroplasma sp. BARC 4886 BAA-1183 Helix nd nd − 0.6 32 Horse fly XXXVI Spiroplasma sp. BARC 4900 BAA-1184 Helix nd nd − 1.0 30 Horse fly XXXVII Spiroplasma sp. BARC 4908 BAA-1187 Helix nd nd − 1.2 32 Horse fly XXXVIII Spiroplasma sp. GSU5450 BAA-1188 Helix nd nd − 1.5 32 Horse fly Nd S. atrichopogonis GNAT3597T BAA-520T (NBRC Helix nd 28 + nd 30 Biting midge 100390T) Nd S. leucomae SMAT BAA-521T (NBRC Helix nd 24 + nd 30 Satin moth 100392T) and, Not determined. bFor descriptions of morphotypes, see text. cGenome size (kbp). dDNA G+C content (mol%). e+, Catabolizes arginine. fDoubling time (h) (Konai et al., 1996a). gOptimum growth temperature (°C). 662 Family II. Spiroplasmataceae group XVI (Abalain-Colloc et al., 1993)] have been divided into Spiroplasma leptinotarsae. ELISA has been used for detection of a total of 15 subgroups. “Serovars” have been defined as geno- Spiroplasma kunkelii (Gordon et al., 1985) and Spiroplasma citri typic clusters varying substantially in metabolism inhibition and (Saillard and Bové, 1983). deformation serology, but that are insufficiently differentiated Optimum growth temperature. Optimal growth tempera- from members of existing groups or subgroups to warrant sepa- tures between 10 and 41°C have been determined (Konai et al., ration. However, with the discovery of a large number of strains 1996a). for some groups (e.g., group VIII), the serovar/subgroup pic- ture has become very confused (Regassa et al., 2004; see Phylog- Substrate metabolism. The ability to ferment glucose and eny, below). produce acid must be examined (Aluotto et al., 1970). The ability to hydrolyze arginine and produce ammonia should be Procedures for species descriptions and minimal standards. assessed (Barile, 1983). See the section on “Biochemical reac- ­Species descriptions of spiroplasmas have been in accord with tions” above for more details. recommendations of minimum standards proposed by the ICSP (International Committee on Systematics of Prokaryotes) Sub- Ecology. The species description must include ecological committee on the Taxonomy of Mollicutes (Brown et al., 2007). information such as isolation site within the host and cultiva- tion conditions, common and binomial host name, geographi- Cloning. Production of spiroplasma lineages produced cal location of host (with GPS), any known interaction between from a single cell or clonings are performed largely by serial the spiroplasma and its host, and, in the case of a pathogen, dilution of filtered cultures using 96-well microtiter plates disease symptoms observed. (Whitcomb et al., 1986; Whitcomb and Hackett, 1987). At a Antibiotic sensitivities. In early studies (Bowyer and Cala- certain dilution, which varies from plating to plating, the mean van, 1974; Liao and Chen, 1981b), spiroplasmas proved to be number of cells per well decreases so that fewer than about 8 of especially sensitive in vitro to tetracycline, erythromycin, tylosin, the 96-wells support growth of a spiroplasma clone. Very prob- tobramycin, and lincomycin. Strains have been isolated that are ably, such clones arise from a single spiroplasmal cell. permanently resistant to kanamycin, neomycin, gentamicin, Cellular morphology. Using dark-field microscopy, cultures erythromycin, and several tetracycline antibiotics (Liao and should appear helical and motile during at least one growth Chen, 1981b). Insensitivity to rifampicin has been studied in phase (see “Morphology” and “Motility” above). However, mor- relation to its inhibition of transcription (see “RNA polymerase phological exceptions do occur (see “Differentiating charac- and spiroplasmal insensitivity to rifampicin” above) and penicil- ters” below and reviewed by Gasparich et al., 2004). lin insensitivity is seen for all spiroplasmas due to the lack of a cell wall. Natural amphipathic peptides such as Gramicidin S 16S rRNA gene sequence analysis. Preliminary identifica- alter the membrane potential of spiroplasma cells and induce tion is performed by PCR amplification using universal 16S the loss of cell motility and helicity (Bévén and Wróblewski, rRNA (Gasparich et al., 2004) or other described primers (e.g., 1997). The toxicity of the lipopeptide antibiotic globomycin was Fukatsu and Nikoh, 1998; Jandhyam, 2008). DNA sequence found to be correlated with an inhibition of spiralin processing analysis using a blast search provides preliminary placement (Bévén et al., 1996). As with Gramicidin S, the antibiotic was within the genus Spiroplasma. Those strains showing close phylo- effective against spiroplasmas, but not Mycoplasma mycoides. Nat- genetic relationships based on 16S rRNA gene sequence analy- ural 18-residue peptaibols (trichorzins PA) are bacteriocidal to ses should then be screened using serological tests. spiroplasmas (Bévén et al., 1998). The mode of action appears Serological tests. The deformation test (Williamson et al., to be permeabilization of the host cell membrane. 1978) is used routinely for serological analyses. Reciprocal titers Hosts, ecology, and pathogenicity of ³320 are generally required for definitive group placement. Deformation is defined as entire or partial loss of helicity. At the Hosts. Almost all spiroplasmas have been found to be asso- end point, cells are often seen in which an unaffected part of the ciated with arthropods or an arthropod connection is strongly helical filament exhibits flexing motility despite the presence of suspected. Hackett et al. (1990) searched for mollicutes in a a bleb on another part of the cell. The deformation titer is the wide variety of insect orders. Isolates were obtained from six reciprocal of the final antiserum dilution that exhibits defor- orders and 14 insect families. Only one of these orders, Odo- mation of ³50% of the cells. Antiserum should be produced nata (dragonflies), was primitive (heterometabolous) and it was for any strain thought to represent a novel serogroup and any speculated that the spiroplasma from a dragonfly host might positive test against characterized groups requires a recriprocal have been acquired via predation. Hackett et al. (1990) sug- test using the newly prepared antiserum. gested that the Spiroplasma/Entomoplasma clade may have arisen The high levels of specificity and sensitivity of the metabo- in a paraneopteran-holometabolan ancestor, coevolved with lism inhibition test make it especially useful for defining groups these orders, and never adapted to more primitive insect orders. and subgroups (Williamson et al., 1979b; Williamson and Whit- Some insect families have an especially rich spiroplasma, ento- comb, 1983). Other serological tests have also been employed moplasma, and mesoplasma flora. for characterization of spiroplasmas. Growth inhibition tests Insect gut. The majority of spiroplasmas appear to be main- were used for delineation of spiroplasma groups I through XI tained in an insect gut/plant surface cycle. Clark (1984) hypoth- (Whitcomb et al., 1982), but were not used thereafter. Growth esized several types of gut infection in which persistence in the inhibition tests are problematic for spiroplasmas because they gut and the ability to invade hemolymph varied among spiro- require development of procedures for obtaining colonies. The plasma species. It has been hypothesized (Hackett and Clark, spiroplasma motility inhibition test (Hackett et al., 1997) has 1989) that the gut cycle was primitive and that other cycles were proved useful for determination of intraspecific variation in derived from it. Spiroplasmas have been isolated from guts of Genus I. Spiroplasma 663 tabanids (Diptera: Tabanidae) worldwide (French et al., 1997, a population of Adalia bipunctata (Sokolova et al., 2002); in sev- 1990, 1996; Jandhyam et al., 2008; Le Goff et al., 1991, 1993; eral strains from the Tucson Drosophila stock culture collection Regassa and Gasparich, 2006; Vazeille-Falcoz et al., 1997; Whit- (Mateos et al., 2006); and in Drosophila melanogaster populations comb et al., 1997a). Examination of diversity trends among the from Uganda and Brazil (Pool et al., 2006). Other organisms tabanid isolates suggests that spiroplasma diversity increases closely associated with their insect hosts were discovered infer- with temperature, resulting in more diversity in southern entially by PCR studies (Fukatsu and Nikoh, 2000, 2001) and climes in the Northern Hemisphere (Whitcomb et al., 2007). also appear to be related to Spiroplasma mirum. They also cause Although evidence points strongly to multiple cycles of hori- preferential male killing in an infected Drosophila population zontal transmission, the sites where such transmission occurs (Anbutsu and Fukatsu, 2003). Natural infection rates of male- remain unknown. However, some tabanids utilize honeydew killing spiroplasmas in Drosophila melanogaster are about 2.3%, (excreta of sucking insects) deposited on leaf surfaces, suggest- as determined for a Brazilian population (Montenegro et al., ing a possible transmission mechanism. Mosquitoes (Chastel 2005), and vary between 0.1 and 3% for Japanese populations and Humphery-Smith, 1991) are also common spiroplasma of Drosophila hydei (Kageyama et al., 2006). The male-killing hosts (Lindh et al., 2005). Additionally, spiroplasmas inhabit the spiroplasma strain isolated from Adalia bipunctata was used to gut of ground beetles (Harpalus pensylvanicus and Anisodactylus artificially infect eight different coccinellid beetle species. The sanctaecrucis) as evidenced by 16S rRNA gene sequence analysis data suggest that host range could serve to limit horizontal of the digestive tract bacterial flora (Lundgren et al., 2007). transfer to closely related host species (Tinsley and Majerus, Plant surfaces. Flowers and other plant surfaces represent a 2007). Supporting this hypothesis was the study that showed major site where spiroplasmas and other microbes are transmit- the interspecific lateral transmission of spiroplasmas from ted from insect to insect (Clark, 1978; Davis, 1978; McCoy et al., Drosophila nebulosa to Drosophila willistoni via ectoparasitic mites 1979). Members of several spiroplasma groups have been iso- (Jaenike et al., 2007). A recent multilocus analysis by Haselkorn lated only from flowers and strains of several other spiroplasmas et al. (2009) showed that Drosophila species are infected with at have been isolated from both insects and flowers. Biological least four distinct spiroplasma haplotypes. evidence suggests that mosquito spiroplasmas are transmitted Studies on Drosophila infections by the sex-ratio organism from insect to insect on flowers (Chastel et al., 1990; Le Goff showed that it did not induce the innate immunity of the insect et al., 1990). It is not known whether any of the so-called “flower (Hurst et al., 2003). The sex-ratio spiroplasmas have been shown spiroplasmas” can exist as true epiphytes. Isolations of spiroplas- to be vertically transmitted through female hosts, with spiroplas- mas from a variety of insects (Clark, 1982; Hackett et al., 1990) mas present during oogenesis (Anbutsu and Fukatsu, 2003). suggest that it is likely that many or most of these flower isolates Although the exact mechanism of male-killing has not been are deposited passively by visiting arthropods. determined, studies have shown that male killing occurs shortly after formation of the host dosage compensation complex (Bent- Plant phloem and sucking insects. Several spiroplasmas pos- ley et al., 2007) and that male Drosophila melanogaster mutants sess a life cycle that involves infection of plant phloem and lacking any of the five genes involved in the dosage compensa- homopterous insects (Bové, 1997; Fletcher et al., 1998; Garnier tion complex are not killed (Veneti et al., 2005). In the Kenyan et al., 2001; Saglio and Whitcomb, 1979). In the course of pas- butterfly Danaus chrysippus, a correlation between male killing sage through the insect, spiroplasmas pass through, accumu- and a recessive allele for a gene controlling infection suscepti- late, or multiply in gut epithelial cells and salivary cells. They bility has been reported. Moreover, infections seemed to have a also accumulate in the insect neurolemma. Large accumula- negative effect on body size (Herren et al., 2007). tions of spiroplasma cells occur frequently in the hemolymph, where they undoubtedly multiply (Whitcomb and Williamson, Ticks. Three Spiroplasma species have been isolated from 1979). Spiroplasmas may multiply in a number of sucking insect ticks. Two of these, Spiroplasma mirum and Spiroplasma sp. 277F, species that have been exposed to diseased plants, but often are from the rabbit tick Haemaphysalis leporispalustris (Tully et al., only a single vector or several vector species transmit spiroplas- 1982; Williamson et al., 1989). The third species was isolated mal pathogens from plant to plant (summarized in Calavan and from Ixodes pacificus ticks and named Spiroplasma ixodetis (Tully Bové, 1989; Whitcomb, 1989; Kersting and Sengonca, 1992). et al., 1995). 16S rRNA gene sequence analysis of spiroplasmas originally isolated from Ixodes ticks and growing in a Buffalo Sex ratio organisms. Once thought to be a genetic factor, the Green Monkey mammalian cell culture line showed a high sex ratio trait in Drosophila was shown by Poulson and Sakaguchi degree of identity with the Spiroplasma ixodetis 16S rRNA gene (1961) to be induced by a micro-organism, Spiroplasma poulsonii (Henning et al., 2006). Analysis of the 16S rRNA gene sequence (Williamson et al., 1999). A number of other spiroplasmas in a from DNA extracted from unfed Ixodes ovatus from Japan indi- variety of insect hosts have been identified that also cause sex cated the presence of spiroplasmas that were also closely related ratio distortions, including isolates from the chrysomelid beetle to Spiroplasma ixodetis (Taroura et al., 2005). The ability of tick Adalia bipunctata (Hurst and Jiggins, 2000; Hurst et al., 1999) spiroplasmas, including Spiroplasma ixodetis, to multiply at 37°C and the butterfly Danaus chrysippus (Jiggins et al., 2000). In reflects the role of vertebrates as tick hosts. The ability of Spiro- addition, 16S rRNA gene sequence analysis identified spiroplas- plasma ixodetis to grow at 32°C as well as 37°C (Tully et al., 1982) mas as the causative agent for male-killing: in a population of may reflect the ecology of some of the cold-blooded vertebrate Harmonia axyridis (ladybird beetle) in Japan (Nakamura et al., hosts of these ticks. There is no evidence that any of these spiro- 2005); in populations of Drosophila neocardini, Drosophila ornati- plasmas are transmitted to vertebrate hosts of the ticks. frons and Drosophila paraguayensis from Brazil (Montenegro et al., 2006, 2005); in populations of Anisosticta novemdecimpunc- Crustaceans. Spiroplasma sp. have recently been isolated in tata (ladybird beetle) in Britain (Tinsley and Majerus, 2006); in both freshwater and salt-water crustaceans. 664 Family II. Spiroplasmataceae

Spiroplasma penaei (strain SHRIMPT) was isolated from the in Drosophila pseudoobscura (Williamson et al., 1989); Spiroplasma hemolymph of Pacific white shrimp (Penaeus vannamei) after penaei in Penaeus vannamei (Nunan et al., 2005); and Spiroplasma high mortalities were observed in an aquaculture pond in ­eriocheiris (Wang et al., 2010) in the Chinese mitten crab, Eriocheir­ Columbia, South America (Nunan et al., 2004). The patho- sinensis (Wang et al., 2004b). Recent studies have focused on genic agent was the spiroplasma (Nunan et al., 2005). Although spiroplasma infection and replication in the midgut and Mal- not cultivated, 16S rRNA gene sequence analysis also revealed pighian tubules of leafhoppers (Özbek et al., 2003). The use the presence of spiroplasmas in the gut of the hydrothermal of immunofluorescence confocal laser scanning microscopy shrimp Rimicaris exoculata (Zbinden and Cambon-Bonavita, has revealed the presence of Spiroplasma kunkelii in the midgut, 2003). In another outbreak, Chinese mitten crab (Eriocheir sin- filter chamber, Malpighian tubules, hindgut, fat tissues, hemo- ensis) reared in aquaculture ponds in China became infected cytes, muscle, trachea, and salivary glands of leafhopper hosts, with tremor disease. The causative agent was determined to be but not in the nerve cells of the brain or nerve ganglia (Ammar a spiroplasma with 99% 16S rRNA gene sequence identity to and Hogenhout., 2005). Plant spiroplasmas may also be patho- Spiroplasma mirum (Wang et al., 2004a, b). However, recent stud- genic for unusual vectors (Whitcomb and Williamson, 1979), ies suggest that the infective agent may be a species similar to, but are much less so for their usual host (Madden and Nault, but distinct from, Spiroplasma mirum (Bi et al., 2008). The same 1983; Nault et al., 1984). In fact, some spiroplasmas are benefi- organism also infects red swamp crayfish (Procambarus clarkii) cial to their leafhopper hosts (Ebbert and Nault, 1994) and it that are co-reared with the Chinese mitten crab (Bi et al., 2008; has been hypothesized that infection plays an important role in Wang et al., 2005) as well as the shrimp Penaeus vannamei (Bi the host’s overwintering strategies (Moya-Raygoza et al., 2007a, et al., 2008). b; Summers et al., 2004). Other hosts. Spiroplasmas have been identified in a variety of Spiroplasma mirum is experimentally pathogenic for a variety other hosts, although not necessarily linked to the gut habitat. of suckling animals, causing cataract and other ocular symp- The first spiroplasma isolated from a lepidopteran came from toms, neural pathology (Clark and Rorke, 1979), and malig- the hemolymph of white satin moth larvae (Leucoma salicis L.) nant transformation in cultured cells (Kotani et al., 1990). from Poland (designated strain SMAT) and was serologically dis- Spiroplasma melliferum also persists and causes pathology in suck- tinct from any previously described spiroplasma group (Oduori ling mice (Chastel et al., 1990, 1991). Spiroplasma eriocheiris is et al., 2005). Another novel spiroplasma (designated strain neurotropic to brain tissue in experimentally injected chicken GNAT3597T) was isolated from biting midges from the genus embryos (Wang et al., 2003). There are two recent reports of Atrichopogon (Koerber et al., 2005). Spiroplasmas that are closely spiroplasmas in aquatic invertebrates. Nunan et al. (2005) char- related to the male-killing spiroplasmas in ladybird beetles acterized a spiroplasma in commercially raised shrimp that led (Majerus et al., 1999; Tinsley and Majerus, 2006) have also been to a lethal disease. Spiroplasma melliferum and Spiroplasma apis identified in the predatory mite Neoseiulus californicus using 16S cause disease in honey bees (Clark, 1977; Mouches et al., 1982a, rRNA gene sequence analysis (Enigl and Schausberger, 2007). 1983a). Intrathoracic inoculation of Spiroplasma taiwanense A broad survey of 16 spider families for the presence of endo- reduced the survival and impaired the flight capacity of inocu- symbionts using 16S rRNA gene sequence analysis revealed lated mosquitoes (Humphery-Smith et al., 1991a), and inocula- that six families contained spiroplasmas, including Agelenidae, tion of Spiroplasma taiwanense per os decreased the survival of Araneidae, Gnaphosidae, Linyphiidae, Lycosidae, and Tetragnathidae mosquito larvae in laboratory trials (Humphery-Smith et al., (Goodacre et al., 2006). 1991b). Spiroplasma poulsonii causes sex ratio abnormalities (male-killing) in Drosophila (Williamson and Poulson, 1979). Biogeography. Spiroplasmas have been identified from hosts Male-killing spiroplasma strains related to Spiroplasma poulsonii in Africa, Asia, Australia, Europe, South America, and North cause necrosis in neuroblastic and fibroblastic cells (Kuroda America. While they are worldwide in distribution, studies sug- et al., 1992). The significance of some biological properties of gest that biodiversity may be greatest in warm climates (Whit- spiroplasmas is incompletely understood. For example, mem- comb et al., 2007). Because spiroplasmas are host-associated, it branes of Spiroplasma monobiae are potent inducers of tumor seems reasonable that Spiroplasma species distribution would be necrosis factor alpha secretion and of blast transformation limited by host biogeography. Early studies indicated that some (Sher et al., 1990a, b) in insect cell culture. spiroplasmas have discrete geographic distributions (Whitcomb Spiroplasmas are implicated by circumstantial evidence, in et al., 1990). As the diversity of sampling sites increases, the the view of some workers, to be associated with human disease. view of spiroplasma biogeography is likely to shift (Regassa and Bastian first claimed in 1979 that spiroplasmas were associ- Gasparich, 2006). Distinct distributions may exist, but probably ated with Creutzfeldt–Jakob Disease (CJD), an extremely rare on a larger geographic scale. While it is not clear what factors scrapie-like disease of humans (Bastian, 1979). Bastian and account for spiroplasma ranges, the level of host specificity and Foster (2001) reported finding spiroplasma 16S rRNA genes in host overwintering ranges may contribute to the biogeography CJD- and scrapie-infected brains that were not observed in con- of Spiroplasma species (Whitcomb et al., 2007). trols. More recent studies (Bastian et al., 2004) presented evi- Pathogenicity. Symptoms of infection and confirmation of dence to show that spiroplasma 16S rRNA genes were found in Koch’s postulates have been reported for the etiologic roles of: brain tissue samples from scrapie-infected sheep, chronic wast- Spiroplasma citri in “stubborn” disease of citrus (Calavan and ing disease-infected cervids, and CJD-infected humans. All the Bové, 1989; Markham et al., 1974); corn stunt spiroplasma brain tissues from non-infected controls were negative for spiro- (Chen and Liao, 1975; Nault and Bradfute, 1979; William- plasmal DNA. These authors further showed that the sequence son and Whitcomb, 1975); Spiroplasma phoeniceum in aster, an of the PCR products from the infected brains was 96% identi- experimental host (Saillard et al., 1987); Spiroplasma poulsonii cal to the Spiroplasma mirum 16S rRNA gene. However, these Genus I. Spiroplasma 665 results could not be replicated in an independent blind study of in turn results in glucose accumulation. Glucose accumulation uninfected and Scrapie-infected hamster brains using the same is known to induce stunting and repression of photosynthesis primers (Alexeeva et al., 2006). A recent study to fulfill Koch’s genes in Arabidopsis thaliana. Such symptoms are precisely those postulate reported the transfer of spiroplasma from transmis- observed in periwinkle plants infected by wild-type Spiroplasma sible spongiform encephalopathy (TSE) brains and Spiroplasma citri (André et al., 2005). mirum to induce spongiform encephalopathy in ruminants Genes that are up- or down-regulated in plants following (Bastian et al., 2007). The current status of the involvement of infection with Spiroplasma citri have been studied by differential spiroplasmas in TSE is the subject of recent reviews (Bastian, display analysis of mRNAs in healthy and symptomatic periwin- 2005; Bastian and Fermin, 2005). Other proposed connections kle plants (Jagoueix-Eveillard et al., 2001). Expression of the between mollicutes and human disease have been evaluated by transketolase gene was inhibited in plants infected by the wild- Baseman and Tully (1997). type spiroplasma, but not by the non-phytopathogenic mutant Mechanism of Spiroplasma citri phytopathogenicity. Transpo- GMT553, further indicating that sugar metabolism and trans- son (Tn4001) mutants have been examined extensively to elu- port are important factors in pathogenicity. Sugar PTS system cidate the molecular mechanisms associated with Spiroplasma permeases have been shown to be important in rapid adapta- citri phytopathogenicity. One of these mutants, GMT553, high- tion to sugar differences between plant host and insect vector lighted the involvement of selective carbohydrate utilization in (André et al., 2003). Spiroplasma citri pathogenicity (see review by Bové et al., 2003). Leafhopper transmission of Spiroplasma citri. Spiroplasmas When introduced into periwinkle plants via injected leafhop- are acquired by leafhopper vectors that imbibe sap from the pers (Circulifer haematoceps), GMT553 multiplied in the plants sieve tubes of infected plants. However, in order to be trans- as actively as wild-type Spiroplasma citri strain GII3, but did not mitted to a plant, the mollicutes need first to multiply in the induce symptoms (Foissac et al., 1997b, c; Gaurivaud et al., insect vector after crossing the gut barrier (Wayadande and 2000b). In this mutant, the transposon was found to be inserted Fletcher, 1995). They multiply to high titers (106–107/ml) in in fruR, a transcriptional activator of the fructose operon (fru- the insect hemolymph, but only when they have reached the RAK; Gaurivaud et al., 2000a). The second gene of the operon, salivary glands can they be inoculated into a plant. One gene fruA, encodes fructose permease, which enables uptake of required for efficient transmission, sc76, was inactivated in a fructose; and the third gene, fruK, encodes 1-phosphofructoki- transposon mutant (G76) with reduced transmissibility (Bou- nase. In mutant GMT553, transcription of the fructose operon tareaud et al., 2004); sc76 encodes a putative lipoprotein. Plants is abolished and, hence, the mutant cannot utilize fructose as infected with the G76 mutant showed symptoms 4–5 weeks a carbon or energy source (Gaurivaud et al., 2000a). Mutant later than those infected with wild-type GII3, but when they GMT553 was functionally complemented for fructose utiliza- appeared, the symptoms induced were severe. Mutant G76 mul- tion and phytopathogenicity in trans by a recombinant fruR– tiplied in plants and leafhoppers as efficiently as the wild-type fruA–fruK operon, fruA–fruK partial operon, or fruA alone, but strain. However, leafhoppers injected with the wild-type spiro- not fruR or fruR–fruA (Gaurivaud et al., 2000a, b). It should be plasma transmitted the spiroplasma to 100% of exposed plants. pointed out that both fructose+ and fructose− spiroplasmas are In contrast, those injected with mutant G76 infected only 50% able to utilize glucose. of the plants. This inefficiency was shown to be associated with Further insight into Spiroplasma citri phytopathogenicity in rela- a numerical decrease in spiroplasma cells in the salivary glands tion to sugar metabolism comes from the production of a spiro- that correlated with reduced output from the stylets of trans- plasma mutant unable to use glucose (André et al., 2005). The mitting leafhoppers; the number of mutant cells transmitted import of glucose into Spiroplasma citri cells involves a phospho- through Parafilm membranes was less than 5% of numbers of transferase (PTS) system composed of two distinct polypeptides wild-type cells transmitted based on colony-forming units. Func- encoded by (1) crr (glucose PTS permease IIAGlc component) tional complementation of the G76 mutant with the sc76 gene and (2) ptsG (glucose PTS permease IICBGlc component). A ptsG restored the wild-type phenotype. Because both wild-type and mutant (GII3-glc1) proved unable to import glucose. When mutant cells multiplied to equally high titers in the hemolymph, introduced into periwinkle (Catharanthus roseus) plants through the results suggest that the mutant is inefficiently passed from leafhopper transmission, the mutant induced severe symptoms the hemolymph into the salivary glands or that it may multiply similar to those obtained with wild-type GII3, in strong contrast to a lower titer in the glands. to the fructose operon mutant, GMT553, which was virtually Transmission of Spiroplasma citri by leafhopper vectors must non-pathogenic. These results indicated that fructose and glu- involve adherence to and invasion of insect host cells. Elec- cose utilization were not equally involved in pathogenicity and tron microscopic studies of leafhopper midgut by Ammar are consistent with biochemical data showing that, in the pres- et al. (2004) have demonstrated the attachment of Spiroplasma ence of both sugars, Spiroplasma citri preferentially used fructose. kunkelii cells by a tip structure to the cell membrane between NMR analyses of carbohydrates in plant extracts revealed the microvilli of epithelial cells. Spiroplasma citri surface protein P89 accumulation of soluble sugars, particularly glucose, in plants was shown to mediate adhesion of the spiroplasma to cells of infected by wild-type Spiroplasma citri GII3 or GII3-glc1, but not the vector Circulifer tenellus and was designated SARP1 (Berg in those infected by GMT553. In the infected plant, Spiroplasma et al., 2001; Yu et al., 2000). The gene encoding SARP1, arp1, citri cells are restricted to the sieve tubes. In the companion cell, was cloned and characterized from Spiroplasma citri BR3-T. The sucrose is cleaved by invertase to fructose and glucose. In the putative gene product SARP1 contains a novel domain at the sieve tube, wild-type Spiroplasma citri cells will use fructose prefer- N terminus, called “sarpin” (Berg et al., 2001). The arp1 gene entially over glucose leading to a decreased fructose concentra- is located on plasmid pBJS-O in Spiroplasma citri (Joshi et al., tion and, consequently, to an increase of invertase activity, which 2005). The Spiroplasma kunkelii plasmid pSKU146 encodes an 666 Family II. Spiroplasmataceae adhesin that is a homolog of SARP1 (Davis et al., 2005). Other ­leptinotarsae (Hackett and Lynn, 1985) were isolated by co- spiroplasma plasmids encode additional adhesin-related pro- cultivation with insect cells. However, the requirement for co- teins. As indicated above (see Plasmids), Spiroplasma citri GII3 cultivation of Spiroplasma leptinotarsae can be circumvented by contains six large plasmids, pSci1 to pSci6 (Saillard et al., placing the primary cultures in BBL anaerobic GasPak jar sys-

2008). Although plasmids pSci1 to pSci5 encode eight differ- tems with low redox potential and enhanced CO2 atmosphere ent Spiroplasma citri adhesin-related proteins (ScARPs), they (Konai et al., 1996b). By lowering the pH of the growth medium are not required for insect transmission (Berho et al., 2006b). from 7.4 to 6.2 and using bromocresol purple as a pH indicator One of the ScARPs, protein P80, shared 63% similarity and (pH 5.2 yellow to pH 6.8 purple), it was possible to perform 45% identity with SARP1. Protein P80 is carried by plasmid metabolism inhibition tests involving Spiroplasma leptinotarsae as pSci4 and has been named ScARP4a. The ScARP-encoding the antigen. The same low-pH medium containing 2.0% Noble genes could not be detected in DNA from non-transmissible agar permitted the growth of colonies (Williamson, unpub- strains (Berho et al., 2006b). Sequence alignments of ScARP lished data). Cohen and Williamson (1988) reported that a proteins revealed that they share common features including fortuitous contamination of H-2 medium by a slow-growing, a conserved signal peptide followed by six to eight repeats of pink-colored yeast (Rhodotorula rubra) permitted primary iso- 39–42 amino acids each, a central conserved region of 330 lation of the non-male-lethal variant of the Dorsophila willistoni amino acids, and a transmembrane domain at the C terminus spiroplasma. After 10–12 passages with yeast, the spiroplasmas (Saillard et al., 2008). were able to grow in yeast-free H-2 medium. Plasmid pSci6 carries the gene for protein P32, which is pres- Maintenance procedures. Adaptation. Most spiroplas- ent in all Spiroplasma citri strains capable of being transmitted by mas can be adapted to a wide variety of media formulations. the leafhopper vector Circulifer haematoceps, but absent from all Spiroplasmas commonly grow more slowly upon transfer to new non-transmissible strains (Killiny et al., 2006). Complementa- media. Initial reduction in growth rate is probably related to tion studies with P32 alone did not restore transmissibility (Kill- a combination of differences in nutrients, pH, osmolality, etc. iny et al., 2006). However, if the pSci6 plasmid was transferred Isolates may grow at only slightly reduced rates during the first to an insect-non-transmissible Spiroplasma citri strain, then the 1–5 passages in a new medium. However, if the new medium phenotype could be converted to insect-transmissible, indicat- is markedly deficient, the growth rate may decrease precipi- ing the likely presence of additional transmissibility factors on tously after 5–10 passages. Continuous careful passaging may pSci6 (Berho et al., 2006a). Indeed, recent data indicates that result in growth rate recovery to levels similar to that in the factors essential for transmissibility are encoded by a 10 kbp initial medium. For such adaptations, best results are achieved fragment of pSci6 (Breton et al., 2010). The finding that the by starting with a 1:1 ratio of old and new media and gradually insect-transmissible strain Spiroplasma citri Alc254 contains only withdrawing the old formulation. Spiroplasma clarkii, after con- a single plasmid, pSci6 (S. Richard and J. Renaudin, unpub- tinuous passage for hundreds of generations, finally adapted to lished) also reinforces the hypothesis that pSci6-encoded deter- extremely simple media (Hackett et al., 1994). Adaptation may minants play a key role in insect transmission of Spiroplasma citri involve mutation and/or activation of adaptive enzymes, or, by its leafhopper vector. possibly, other mechanisms. Growth rates in such simple media Enrichment and isolation procedures were much slower than those in rich media. Maintenance media. Spiroplasma citri can be cultivated in Isolation. Success in the isolation of fastidious spiroplasmas is a relatively simple medium that utilizes sorbitol to maintain influenced strongly by the titer of the inoculum. Spiroplasmas osmolality (Saglio et al., 1971). A modification of this medium have been isolated from salivary glands, gut, and nerve tissues (BSR) has been used extensively for Spiroplasma citri (Bové and of their insect hosts. Many spiroplasmas envisioned by dark- Saillard, 1979), in which the horse serum content was lowered field microscopy have proved to be noncultivable (Hackett and to 10% and the fresh yeast extract was omitted. Other simple Clark, 1989). Initial insect extracts in growth media are passed media, such as C-3G (Liao and Chen, 1977), are suitable for through a 0.45 mm filter. The filtrate is then observed daily for maintenance or large-batch cultivation of fast-growing spiro- pH indicator change. An alternative to filtration involves the use plasmas. This medium is also adequate for primary isolation of of antibiotics or other inhibitors (Grulet et al., 1993; Markham Spiroplasma kunkelii (Alivizatos, 1988). However, cultivation of et al., 1983; Whitcomb et al., 1973). Spiroplasma isolations from more fastidious spiroplasmas is best achieved in M1D medium infected plants are best obtained from sap expressed from vas- (Hackett and Whitcomb, 1995; Whitcomb, 1983; Williamson cular bundles of hosts showing early disease symptoms. Plant and Whitcomb, 1975) if they derive from plant or insect habi- sap often contains spiroplasmal substances (Liao et al., 1979) tats. SP-4 medium (Tully et al., 1977) is very suitable if spiroplas- whose presence in primary cultures may necessitate blind pas- mas derive from tick habitats. SM-1 medium (Clark, 1982) has sage or serial dilution. also been successfully employed for many insect spiroplasmas. Isolation media. M1D medium (Whitcomb, 1983) has been Defined media. Spiroplasma floricola and some strains of used for primary isolations of the large proportion of spiro- Spiroplasma apis have been cultivated in chemically defined plasma species. SP-4 medium, a rich formulation derived from media (Chang, 1989, 1982). experiments with M1D, is necessary for isolation of Spiroplasma mirum from fluids of the embryonated egg (Tully et al., 1982). Preservation. Spiroplasmas are routinely preserved by SP-4 medium is also required for isolation of Spiroplasma ixode- lyophilization (FAO/WHO, 1974). Most spiroplasmas can be tis (Tully et al., 1981). Some very fastidious spiroplasmas such maintained at −70°C indefinitely. Preservation success at −20°C as Spiroplasma poulsonii (Hackett et al., 1986) and Spiroplasma­ is irregular and uncertain. Genus I. Spiroplasma 667

Differentiation of the genus Spiroplasma and Davis, 1980; Liao and Chen, 1981a; Rahimian and Gumpf, from other closely related taxa 1980). Given these challenges, an alternative method was identi- fied in serology. Surface serology of spiroplasmas has proven to Spiroplasmas can be clearly differentiated from all other micro- be a robust surrogate for DNA–DNA hybridization assays. organisms by their unique properties of helicity and motility, combined with the complete absence of periplasmic fibrils, Phylogeny. Phylogenetic studies of Spiroplasma became cell walls, or cell wall precursors. However, spiroplasmas may possible when Carl Woese and colleagues, searching for a be nonhelical under some environmental conditions or when molecular chronometer by which microbial evolution could be cultures are in the stationary phase of growth. Morphological reconstructed, found that rRNA met most or all of the desired study of the organisms in the exponential phase of growth usu- criteria (reviewed by Woese, 1987). Today, sequencing of rRNA ally reveals characteristic helical forms. However, the existence genes has become a universal tool for phylogenetic reconstruc- of spiroplasmas that appear entirely or largely as nonhelical tion. Early phylogenetic analyses involved distance estimates forms (e.g., Spiroplasma ixodetis and group XXIII strain TIUS- (DeSoete, 1983). Later, neighbor-joining (Saitou and Nei, 1987) 1) raises the theoretical possibility that an organism situated was introduced into mollicute phylogeny (Maniloff, 1992) at an apomorphic (advanced) position on the spiroplasma and several mollicute workers have used maximum-likelihood phylogenetic tree could totally lack helicity or motility. In fact, (Felsenstein, 1993). The extensive and classical studies of K.-E. the clade containing Mycoplasma mycoides and the Entomoplas- Johansson’s group (Johansson et al., 1998; Pettersson et al., mataceae has apparently done exactly that. Spiroplasma floricola 2000) were completed using neighbor-joining, but selectively produces nonhelical, but viable, cells early in stationary phase, confirmed by maximum-likelihood and maximum-parsimony which can begin within 24 h of medium inoculation. For rea- (Swofford, 1998). Gasparich et al. (2004) studied the phylogeny sons such as this, it is necessary to examine cultures throughout of Spiroplasma and its nonhelical descendants using parsimony, the growth cycle to ensure that an adequate search for helical maximum-likelihood, distance, and neighbor-joining analyses, cells has been made. which generated 24 phylogenetic inferences that were com- mon to all, or almost all, of the trees. More recently, Bayesian Taxonomic comments analysis [MrBayes (http://mrbayes.csit.fsu.edu/index.php)] was used to examine an expanded Spiroplasma Apis clade based Early history. The term “spiroplasma” was first coined as a on 16S rRNA and 16S–23S ITS sequences; the analyses showed trivial term to describe helical organisms shown to be associ- congruency between Bayesian and maximum-parsimony trees ated with corn stunt disease (Davis et al., 1972a, b) that could (Jandhyam et al., 2008). not, at that time, be cultivated (Davis and Worley, 1973). Shortly Woese et al. (1980) presented a 16S rRNA gene-based phylo- thereafter, when similar organisms associated with citrus stub- genetic tree for Mollicutes, including Spiroplasma, indicating that born disease were characterized (Saglio et al., 1973), the triv- these wall-less bacteria were related to members of the phylum ial term was adopted as the generic name and the stubborn Firmicutes such as Lactobacillus spp. and Clostridium innocuum. organism was named Spiroplasma citri. This species was the The tree suggested that Mollicutes might be monophyletic. first cultured spiroplasma and the first cultured mollicute of However, a later study by Weisburg et al. (1989) with 40 addi- plant origin. Shortly after the stubborn agent was named, the tional species of Mollicutes including ten spiroplasmas, failed genus Spiroplasma was elevated to the status of a family (Skri- to confirm the monophyly of Mollicutes at the deepest branch- pal, 1974) and added to the Approved Lists of Bacterial Names ing orders. The Woese et al. (1980) model also suggested that (Skripal, 1983). The organism that was eventually named Spiro- the genus Mycoplasma might not be monophyletic, in that the plasma mirum (Tully et al., 1982) was isolated by Clark (1964) type species, Mycoplasma mycoides, and two related species, Myco- in embryonated chicken eggs soon after the discovery of the plasma capricolum and Mycoplasma putrefaciens, appeared to be organism later named Spiroplasma poulsonii. Because Spiroplasma more closely related to the Apis clade of Spiroplasma than to mirum readily passed through filters, it was first mistaken for the other Mycoplasma species. This conclusion was supported a virus. The subgroup I-4 277F spiroplasma was cultivated in by analyses of the 5S rRNA genes (Rogers et al., 1985). All trees 1968, but was mistaken for a spirochete (Pickens et al., 1968). so far obtained indicate that the acholeplasma-anaeroplasma The first organism to be initially recognized as a spiroplasma (Acholeplasmatales–Anaeroplasmatales) and spiroplasma-myco- was Spiroplasma kunkelii, which was envisioned by dark-field and plasma (Mycoplasmatales–Entomoplasmatales) lineages are mono- electron microscopy in 1971–1972 and cultivated in 1975 (Liao phyletic, but are separated by an ancient divergence. and Chen, 1977; Williamson and Whitcomb, 1975). More than In-depth analysis of characterized spiroplasmas and their non- a decade passed before Clark (1982) showed that spiroplasmas, helical descendants indicates the existence of four major clades many of them fast-growing, occurred principally in insects. within the monophyletic spiroplasma-mycoplasma lineage (Gas- Species concept. The species concept in spiroplasmas, as in parich et al., 2004; Figure 113). One of the four clades consists all bacteria, was based on DNA–DNA reassociation (ICSB Sub- of the nonhelical species of the mycoides group (as defined committee on the Taxonomy of Mollicutes, 1995; Johnson, 1994; by Johansson, 2002) as well as the six species of Entomoplasma Rosselló-Mora and Amann, 2001; Stackebrandt et al., 2002; and twelve species of Mesoplasma (the Entomoplasmataceae); this Wayne et al., 1987). In practice, DNA–DNA reassociation results assemblage was designated the Mycoides-Entomoplasmataceae with spiroplasmas have proven difficult to standardize. Estimates clade. The analyses indicated that the remaining three clades of reassociation between Spiroplasma citri (subgroup I-1) and represented Spiroplasma species. One of these clades, the Apis Spiroplasma kunkelii (subgroup I-3) varied between 30 and 70%, clade, was found to be a sister to the Mycoides-Entomoplasma- depending on the method employed and the degree of strin- taceae clade. The Apis clade contains a large number of spe- gency (Bové and Saillard, 1979; Christiansen et al., 1979; Lee cies from diverse insect hosts, many of which possess life cycles Spiroplasma chrysopicola * Spiroplasma syrphidicola Spiroplasma sp. TAAS-1 Spiroplasma mirum Spiroplasma sp. LB-12 Spiroplasma sp. 277F Spiroplasma sp. N525 Spiroplasma poulsonii * Spiroplasma penaei Spiroplasma insolitum Spiroplasma phoeniceum P40 Spiroplasma kunkelii CR2-3x * * Spiroplasma citri Spiroplasma melliferum Entomoplasma freundtii * Mycoplasma mycoides Mesoplasma seiffertii Spiroplasma monobiae * Spiroplasma diabroticae * Spiroplasma floricola Spiroplasma sp. BIUS-1 Spiroplasma sp. W115 * Spiroplasma cantharicola CC-1 * Spiroplasma sp. CB-1 Spiroplasma sp. Ar-1357 * Spiroplasma diminutum Spiroplasma taiwanense Spiroplasma gladiatoris * Spiroplasma lineolae TALS-2 Spiroplasma sp. BARC 1901 * Spiroplasma helicoides Spiroplasma clarkii * Spiroplasma apis * Spiroplasma montanense * Spiroplasma litorale * Spiroplasma turonicum Spiroplasma corruscae Spiroplasma culicicola Spiroplasma velocicrescens Spiroplasma chinense Spiroplasma leptinotarsae * Spiroplasma lampyridicola Spiroplasma sabaudiense Spiroplasma alleghenense Spiroplasma ixodetis Mycoplasma pneumoniae Ureaplasma urealyticum Acholeplasma laidlawii ’Candidatus Phytoplasma’ sp. vigna Il Anaeroplasma bactoclasticum Clostridium innocuum * Bacillus subtilis TB11 Asteroleplasma anaerobium Escherichia coli

Scale: 0.1 substitutions/site

FIGURE 113. Phylogenetic relationships of members of the class Mollicutes and selected members of the phylum Firmicutes. The phylogram was based on a Jukes-Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Escherichia coli was the outgroup. Bootstrap values (100 replicates) <50% are indicated (*). The GenBank accession numbers for 16S rRNA gene sequences used are: Mycoplasma mycoides (U26039); Mycoplasma pneumoniae (M29061); Entomoplasma freundtii (AF036954); Mesoplasma seiffertii (AY351331); Spiroplasma apis (M23937); Spiroplasma clarkii (M 24474); Spiroplasma gladiatoris (M24475); Spiroplasma taiwan- ense (M24476); Spiroplasma monobiae (M24481); Spiroplasma diabroticae (M24482); Spiroplasma melliferum (AY325304); Spiroplasma citri (M23942); Spiroplasma mirum (M24662); Spiroplasma ixodetis (M24477); Spiroplasma sp. strain N525 (DQ186642); Spiroplasma poulsonii (M24483); Spiroplasma penaei (AY771927); Spiroplasma phoeniceum (AY772395); Spiroplasma kunkelii (DQ319068); Spiroplasma cantharicola (DQ861914); Spiroplasma lineolae (DQ860100); Spiroplasma sp. strain 277F (AY189312); Spiroplasma sp. strain LB-12 (AY189313); Spiroplasma insolitum (AY189133); Spiroplasma flori- cola (AY189131); Spiroplasma syrphidicola (AY189309); Spiroplasma chrysopicola (AY189127); Spiroplasma sp. strain TAAS-1 (AY189314); Spiroplasma culicicola (AY189129); Spiroplasma velocicrescens (AY189311); Spiroplasma sabaudiense (AY189308); Spiroplasma corruscae (AY189128); Spiroplasma sp. strain CB-1 (AY189315); Spiroplasma sp. strain Ar-1357 (AY189316); Spiroplasma turonicum (AY189310); Spiroplasma litorale (AY189306); Spiroplasma lampyridicola (AY189134); Spiroplasma leptinotarsae (AY189305); Spiroplasma sp. strain W115 (AY189317); Spiroplasma chinense (AY189126); Spiro- plasma diminutum (AY189130); Spiroplasma alleghenense (AY189125); Spiroplasma sp. strain BIUS-1 (AY189319); Spiroplasma montanense (AY189307); Spiroplasma helicoides (AY189132); Spiroplasma sp. strain BARC 1901 (AY189320); Ureaplasma urealyticum (M23935); “Candidatus Phytoplasma” sp. Vigna II (AJ289195); Acholeplasma laidlawii (M23932); Anaeroplasma bactoclasticum (M25049); Clostridium innocuum (M23732); Asteroleplasma anaero- bium (M22351); Bacillus subtilis (AF058766); Escherichia coli (J01859). Genus I. Spiroplasma 669 involving transmission between the guts of insects and plant existing subgroups (Regassa et al., 2004). Over time, the con- surfaces. One of these species, Spiroplasma sp. TIUS-1 (group cept of the microbial species has undergone a subtle change. XXVIII) has very poor helicity and a genome size of 840 kbp, It is now recognized (Rosselló-Mora and Amann, 2001; Stack- smaller than that of most other spiroplasmas. This species ebrandt et al., 2002) that microbial species must at times consist diverged from the spiroplasma lineage close to the node of of strain clusters that may contain species with <70% similarity entomoplasmal divergence and can be envisioned as a “missing as determined by DNA–DNA reassociation. Group VIII spiro- link” in the evolutionary development of the Mycoides-Ento- plasmas may comprise such a cluster and efforts to subdivide moplasmataceae clade. The other two Spiroplasma clades are the this cluster may have been inadvisable. monospecific Ixodetis clade (group VI) and the Citri-Chrysopi- Character mapping of non-genetic features has been com- cola-Mirum clade (with representatives from groups I, II, V, pleted in conjunction with phylogenetic analyses (Gasparich and VIII). The Citri-Chrysopicola-Mirum clade contains Spiro- et al., 2004). Serological classifications of spiroplasmas are gen- plasma mirum, Spiroplasma poulsonii, the three subgroups of the erally supported by the trees, but the resolution of genetic anal- Chrysopicola (group VIII) clade, and the nine subgroups of the yses appears to be much greater than that of serology. Genome Citri (group I) clade. Members of group I and group VIII show size and G+C content were moderately conserved among closely close intragroup relationships, as indicated by the similarities of related strains. Apparent conservation of slower growth rates their 16S rRNA gene sequences (Gasparich et al., 2004). DNA– in some clades was most likely attributable to host affiliation; DNA reassociation studies for group I (Bové et al., 1983, 1982; spiroplasmas of all groups that were well adapted to a specific Junca et al., 1980) spiroplasmas supported the subgroup clus- host had slower growth rates. Sterol requirements were poly- ter. The Chrysopicola clade (group VIII) subgroups have met a phyletic, as was the ability to grow in the presence of PES, but different fate. Although their DNA–DNA similarities in reasso- not serum. ciation procedures were slightly less than 70%, their 16S rRNA gene sequence similarities were >99% (Gasparich et al., 1993c). Acknowledgements The strains of this group, including not only the subgroups, but We gratefully acknowledge J. Dennis Pollack for assistance on a plethora of isolates from the same ecological context, appear sections concerning intermediary metabolism. to form a matrix of interrelated strains. Boundaries that seemed Further reading clear when the subgroups were initially described, eventually eroded beyond recognition. The 16S rRNA gene sequence sim- Whitcomb, R.F. and J.G. Tully (editors). 1989. The Mycoplas- ilarities are too high to permit cladistic analysis and even 16S– mas, vol. 5, Spiroplasmas, acholeplasmas, and mycoplasmas 23S rRNA spacer region sequence analysis failed to resolve the of plants and arthropods. Academic Press, New York.

List of species of the genus Spiroplasma 1. Spiroplasma citri Saglio, L’Hospital, Laflèche, Dupont, Source: isolated from leaves, seed coats, and fruits of Bové, Tully and Freundt 1973, 202AL ­citrus plants (orange and grapefruit) infected with stub- cit¢ri. L. masc. n. citrus the citrus; N.L. masc. n. Citrus generic born disease, and from other naturally infected plants (e.g., name; N.L. gen. n. citri of Citrus, to denote the plant host. periwinkle, horseradish or brassicaceous weeds) or insects. Cells are helices that divide in mid-exponential phase Known from Mediterranean and other warm climates of when they have four turns. Helical filaments are usually Europe, North Africa, Near and Middle East, and the West- 100–200 nm in diameter and 2–4 mm in length. Cells ern United States (California and Arizona). DNA G+C content (mol%): 25–27 (T , Bd). are longer in late exponential phase and early stationary m phase. Nonviable cells in late exponential phase are non- Type strain: ATCC 27556, Morocco strain, R8-A2. helical. Sequence accession no. (16S rRNA gene): M23942. Colonies on solid media containing 20% horse serum 2. Spiroplasma alleghenense Adams, Whitcomb, Tully, Clark, and 0.8% Noble agar (Difco) are umbonate, 60–150 mm in Rose, Carle, Konai, Bové, Henegar and Williamson 1997, diameter. Moderate turbidity is produced in liquid cultures. 762VP Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, al.le.ghen.en¢se. N.L. neut. adj. alleghenense of the Allegheny groups, and subgroups, but shares some cross-reactivity Mountains, referring to the geographic origin of the type with members of other group I subgroups. Has close phy- strain, the range of the Appalachian Mountains from which logenetic affinities with other group I members, and with it was derived. Spiroplasma poulsonii in trees constructed using 16S rRNA Cells are motile helical filaments, 100–300 nm in gene sequences. ­diameter. Under many growth conditions, cells in medium Pathogenic for citrus plants and a variety of plant hosts are deformed. Colonies on solid medium containing (aster, periwinkle, broad bean) following transmission by 3.0% Noble agar are small and granular and never have infected insects (leafhoppers). a ­fried-egg appearance. Biological properties are listed in DNA–DNA renaturation experiments confirm serologi- Table 142. cal data that indicate that the differences between the type Serologically distinct from other Spiroplasma species, strain (subgroup I-1) and other subgroups of group I are groups, and subgroups. When tested as an antigen, cross- great enough to warrant its designation as a distinct species. reacts broadly with many nonspecific sera (one-way reac- The genome size is 1820 kbp (PFGE). tion). Has close phylogenetic relationship to Spiroplasma 670 Family II. Spiroplasmataceae

sabaudiense (group XIII) and strain TIUS-1 (group XXVIII) Source: isolated from a pooled sample of two nearly iden- in trees constructed using 16S rRNA gene sequences. The tical species of biting midges (Atrichopogon geminus and genome size is 1,465 kbp (PFGE). Atrichopogon levis).

Source: isolated from the hemolymph of a common scor- DNA G+C content (mol%): 28.8 ± 1 (Tm). pion fly,Panorpa helena in West Virginia, USA. Type strain: ATTC BAA-520, NBRC 100390, GNAT3597.

DNA G+C content (mol%): 31 ± 1 (Tm, Bd). Sequence accession no. (16S rRNA gene): not available. Type strain: ATCC 51752, PLHS-1. Sequence accession no. (16S rRNA gene): AY189125. 5. Spiroplasma cantharicola Whitcomb, Chastel, Abalain- Colloc, Stevens, Tully, Rose, Carle, Bové, Henegar, Hackett, 3. Spiroplasma apis Mouches, Bové, Tully, Rose, McCoy, Carle- Clark, Konai and Williamson 1993a, 423VP Junca, Garnier and Saillard 1984b, 91VP (Effective publica- can.thar.i¢co.la. Gr. kantharos scarab beetle; L. suff. -cola tion: Mouches, Bové, Tully, Rose, McCoy, Carle-Junca, Gar- (from L. n. incola) inhabitant, dweller; N.L. n. cantharicola nier and Saillard 1983a, 383.) an inhabitant of a family of beetles. a¢pis. L. fem. n. apis, -is a bee, and also the genus name of The morphology is as described for the genus. Cells are the honey bee, Apis mellifera; L. gen. n. apis of a bee, of Apis helical and motile. Colonies on solid medium containing mellifera, the insect host for this species. 0.8% Noble agar are diffuse, without fried-egg morphology. The morphology is as described for the genus. Helical Biological properties are listed in Table 142. filaments are usually 100–150 nm in diameter and 3–10m m Serologically distinct from other Spiroplasma species, in length. Colonies on solid medium containing 20% fetal groups, and subgroups, but shares some reciprocal cross- bovine serum and 0.8% Noble agar (Difco) are usually dif- reactivity with members of other group XVI subgroups. fuse, rarely exhibiting central zones of growth into the agar. Not yet classified phylogenetically, but no doubt closely Colonies on solid medium with 2.25% Noble agar and 1–5% related to subgroups XVI-2 and XVI-3, which are sisters form- bovine serum fraction are smaller, but exhibit central zones ing a clade related to Spiroplasma diminutum in phylogenetic of growth into the agar and some peripheral growth on the trees constructed using 16S rRNA gene sequences. More- agar surface around the central zones. Marked turbidity is over, DNA–DNA renaturation experiments confirm that the produced during growth in most spiroplasma media (BSR, differences between the type strain and other subgroups of M1A, SP-4). Biological properties are listed in Table 142. group XVI are great enough to warrant its designation as a Serologically distinct from other Spiroplasma species, distinct species. The genome size is 1320 kbp (PFGE). groups, and subgroups. Many strains show partial cross- Source: isolated from the gut of an adult cantharid beetle reactions when tested against sera to strain B31T (Tully (Cantharis carolinus) in Maryland, USA. Based on its resi- et al., 1980). These strains show more than 80% DNA–DNA dence in the gut of a flower-visiting insect, this species is reassociation with strain B31T, but their exact taxonomic thought to be transmitted on flowers. status is unclear. Some strains show a very low level recipro- DNA G+C content (mol%): 26 ± 1 (Tm, Bd, HPLC). cal cross-reaction with Spiroplasma montanense in deforma- Type strain: ATCC 43207, CC-1. tion serology. In accordance with serology, Spiroplasma apis Sequence accession no. (16S rRNA gene): DQ861914. and Spiroplasma montanense are sister species in phylogenetic trees constructed using 16S rRNA gene sequences. The 6. Spiroplasma chinense Guo, Chen, Whitcomb, Rose, Tully, genome size is 1300 kbp (PFGE). Williamson, Ye and Chen 1990, 424VP Etiologic agent of May disease of honey bees in south- chi.nen¢se. N.L. neut. adj. chinense of China, the location western France. Various strains of the organism exhibit where the organism was first isolated. experimental pathogenicity for young honey bees in feed- The morphology is as described for the genus. Cells are ing experiments. motile helical filaments ~160 nm in diameter. Colonies on Source: isolated from honey bees (Apis mellifera) and from solid medium containing 0.8–1.0% Noble agar are diffuse flower surfaces in widely separated geographic regions with many small satellite colonies; growth on 2.25% agar (France, Corsica, Morocco, USA). produces smaller rough or granular colonies and fewer sat- DNA G+C content (mol%): 29–31 (T , Bd). m ellite forms. Biological properties are listed in Table 142. Type strain: ATCC 33834, B31. Serologically distinct from other Spiroplasma species, Sequence accession no. (16S rRNA gene): AY736030. groups, and subgroups. Phylogenetically, this species is 4. Spiroplasma atrichopogonis Koerber, Gasparich, Frana and closely related to Spiroplasma velocicrescens in phylogenetic Grogan 2005, 291VP trees constructed using 16S rRNA gene sequences. The a.tri.cho.po.go¢nis. N.L. gen. n. atrichopogonis of Atrichopogon, genome size is 1530 kbp (PFGE). systematic genus name of a biting midge (Diptera: Cer- Source: isolated from flower surfaces of bindweed Calyste( - atopogonidae). gia hederacea) in Jiangsu, People’s Republic of China. DNA G+C content (mol%): 29 ± 1 (Tm). The morphology is as described for the genus. Cells are Type strain: ATCC 43960, CCH. helical and motile. Biological properties are listed in Table Sequence accession no. (16S rRNA gene): AY189126. 142. Serologically distinct from previously established Spiro- 7. Spiroplasma chrysopicola Whitcomb, French, Tully, Gas- plasma species, groups, and subgroups. The genome size parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, has not been determined. Clark and Williamson 1997b, 718VP Genus I. Spiroplasma 671

chry.so.pi¢co.la. N.L. n. Chrysops a genus of deer flies in 2.25% Noble agar are slightly diffuse to discrete and gen- the Tabanidae; L. suff. -cola (from L. n. incola) inhabitant, erally without the characteristic fried-egg morphology. Bio- dweller; N.L. n. chrysopicola inhabiting Chrysops spp. logical properties are listed in Table 142. Helical motile filaments are short and thin, passing a 220 Serologically distinct from previously established Spiro- nm filter quantitatively. Grows to titers as high as 1011/ml. plasma species, groups, and subgroups. Phylogenetically, Colonies on solid medium containing 2.25% Noble agar closely related to Spiroplasma turonicum and Spiroplasma lito- have dense centers and smooth edges (a fried-egg appear- rale in trees constructed using 16S rRNA gene sequences. ance) and do not have satellites. Biological properties are The genome size has not been determined. listed in Table 142. Source: isolated from the gut of an adult lampyrid beetle Serologically distinct from other Spiroplasma species, (Ellychnia corrusca) in Maryland in early spring, but found groups, and subgroups, but exhibits some reciprocal or much more frequently in horse flies in summer months. one-way cross-reactivity with members of other group VIII Other strains have been collected from Canada and Geor- subgroups. Some strains of group VIII spiroplasmas may gia, Connecticut, South Dakota, and Texas, USA. DNA G+C content (mol%): 26 ± 1 (T , Bd). be difficult to identify to subgroup. Shares less than 70% m DNA–DNA reassociation with Spiroplasma syrphidicola and Type strain: ATCC 43212, EC-1. strain TAAS-1 (subgroup VIII-3). Phylogenetically, this spe- Sequence accession no. (16S rRNA gene): AY189128. cies is closely related to other group VIII strains in trees 10. Spiroplasma culicicola Hung, Chen, Whitcomb, Tully and constructed using 16S rRNA gene sequences. The 16S Chen 1987, 368VP rRNA gene similarity coefficients of group VIII spiroplas- mas are >0.99, so this gene is insufficient for distinguishing cu.li.ci′co.la. L. n. culex, -icis a gnat, midge, and also a genus species in group VIII. The genome size is 1270 kbp (PFGE). of mosquitoes (Culex, family Culicidae); L. suffix -cola (from Pathogenicity for insects has not been determined. L. n. incola) inhabitant, dweller; N.L. n. culicicola intended Source: isolated from the gut of a deer fly Chrysops( sp.) to mean an inhabitant of the Culicidae. in Maryland, USA. Other strains from deer flies have been Cells are pleomorphic, but are commonly very short collected from as far west as Wyoming, from New England, motile helices, 1–2 mm in length. Colonies on solid medium and very rarely, as far south as Georgia, USA. containing 1% Noble agar have a fried-egg appearance with DNA G+C content (mol%): 30 ± 1 (Bd). satellites. Biological properties are listed in Table 142. Type strain: ATCC 43209, DF-1. Serologically distinct from other Spiroplasma species, Sequence accession no. (16S rRNA gene): AY189127. groups, and subgroups. Phylogenetically, this species is 8. Spiroplasma clarkii Whitcomb, Vignault, Tully, Rose, Car- placed in the classical Apis cluster of spiroplasmas, but does le, Bové, Hackett, Henegar, Konai and Williamson 1993c, not have an especially close neighbor in trees constructed 264VP using 16S rRNA gene sequences. The genome size is 1350 kbp (PFGE). clar¢ki.i. N.L. masc. gen. n. clarkii of Clark, in honor of Tru- Source: isolated from a triturate of a salt marsh mosquito man B. Clark, a pioneer spiroplasma ecologist. (Aedes sollicitans) collected in New Jersey, USA. The morphology is as described for the genus. The heli- DNA G+C content (mol%): 26 ± 1 (Tm, Bd). cal motile filaments remain stable throughout exponential Type strain: ATCC 35112, AES-1. growth. Colonies on solid medium containing 0.8% Noble Sequence accession no. (16S rRNA gene): AY189129. agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142. 11. Spiroplasma diabroticae Carle, Whitcomb, Hackett, Tully, Serologically distinct from other Spiroplasma species, groups, Rose, Bové, Henegar, Konai and Williamson 1997, 80VP and subgroups. Phylogenetically, this species is placed in the di.a.bro.ti′cae. N.L. gen. n. diabroticae of Diabrotica, referring classical Apis cluster of spiroplasmas, but it does not have an to Diabrotica undecimpunctata, the chrysomelid beetle from especially close neighbor in trees constructed using 16S rRNA which the organism was isolated. gene sequences. The genome size is 1720 kbp (PFGE). Patho- genicity for insects has not been determined. The morphology is as described for the genus. Cells are Source: isolated from the gut of a larval scarabaeid beetle helical, motile filaments, 200–300 nm in diameter. Colonies (Cotinus nitida) in Maryland, USA. on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are DNA G+C content (mol%): 29 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 33827, CN-5. listed in Table 142. Sequence accession no. (16S rRNA gene): M24474. Serologically distinct from other established Spiroplasma species, groups, and subgroups. Phylogenetically, closely 9. Spiroplasma corruscae Hackett, Whitcomb, French, Tully, related to Spiroplasma floricola in trees constructed using Gasparich, Rose, Carle, Bové, Henegar, Clark, Konai, Clark 16S rRNA gene sequences. The genome size is 1350 kbp and Williamson 1996c, 949VP (PFGE). cor.rus¢cae. N.L. gen. n. corruscae of corrusca, referring to the Source: isolated from the hemolymph of an adult chry- species of firefly beetle (Ellychnia corrusca) from which the somelid beetle, Diabroticae undecimpunctata howardi. organism was first isolated. DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). The morphology is as described for the genus. Cells are Type strain: ATCC 43210, DU-1. helical and motile. Colonies on solid medium containing Sequence accession no. (16S rRNA gene): M24482. 672 Family II. Spiroplasmataceae

12. Spiroplasma diminutum Williamson, Tully, Rosen, Rose, common to several spiroplasmal inhabitants of horse flies, Whitcomb, Abalain-Colloc, Carle, Bové and Smyth 1996, that confers a high level of one way cross-reactivity when it 232VP is used as an antigen. Phylogenetically, closely related to two di.min.u¢tum. L. v. deminuere to break into small pieces, other tabanid spiroplasmas, Spiroplasma helicoides and group make smaller; L. neut. part. adj. diminutum made smaller, XXXIV strain B1901, in phylogenetic trees constructed reflecting a smaller size. using 16S rRNA gene sequences. The genome size has not been determined. The morphology is as described for the genus. Cells are Source: isolated from the gut of a horse fly Tabanus( gladi- short (1–2 mm), helical filaments, 100–200 nm in diameter ator) in Maryland, USA. Other strains have been collected that appear to be rapidly moving, irregularly spherical bod- at various locations in the southeastern United States. ies when exponential phase broth cultures are examined DNA G+C content (mol%): 26 ± 1 (Bd). under dark-field illumination. Colonies on solid medium Type strain: ATCC 43525, TG-1. containing 1.6% Noble agar have dense centers, granular Sequence accession no. (16S rRNA gene): M24475. perimeters, and nondistinct edges with satellite colonies. Biological properties are listed in Table 142. 15. Spiroplasma helicoides Whitcomb, French, Tully, Gas- Serologically distinct from other Spiroplasma species, parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, groups, and subgroups. Phylogenetically, closely related Clark and Williamson 1997b, 718VP to group XVI spiroplasmas in trees constructed using he.li.co.i¢des. Gr. n. helix spiral; Gr. suff. -oides like, resem- 16S rRNA gene sequences. The genome size is 1080 kbp bling, similar; N.L. neut. adj. helicoides spiral-like. (PFGE). The morphology is as described for the genus. Cells are Source: isolated from a frozen triturate of adult female motile helical filaments that lack a cell wall. Colonies on Culex annulus mosquitoes collected in Taishan, Taiwan. solid medium containing 2.25% Noble agar have dense DNA G+C content (mol%): 26 ± 1 (T , Bd, HPLC). m centers and smooth edges, do not have satellites, and have Type strain: ATCC 49235, CUAS-1. a perfect fried-egg appearance. Biological properties are Sequence accession no. (16S rRNA gene): AY189130. listed in Table 142. 13. Spiroplasma floricola Davis, Lee and Worley 1981, 462VP Serologically distinct from other Spiroplasma species, flor.i¢co.la. L. n. flos, -oris a flower; L. suff. -cola (from L. n. groups, and subgroups. This species has a specific antigen, incola) inhabitant, dweller; N.L. n. floricola flower-dweller. common to several spiroplasmal inhabitants of horse flies, that confers a high level one-way cross-reaction when it is The morphology is as described for the genus. Helical used as antigen. Phylogenetically, closely related to two cells are 150–200 nm in diameter and 2–5 mm in length. other tabanid spiroplasmas, Spiroplasma gladiatoris and Spiro- Colonies on solid media have granular central regions plasma sp. BARC 1901, in trees constructed using 16S rRNA surrounded by satellite colonies that probably form after gene sequences. Genome size has not been determined. migration of cells from the central focus. Biological proper- Source: isolated from the gut of a horse flyTabanus abactor ties are listed in Table 142. collected in Oklahoma, USA. Other strains have been col- Serologically distinct from other Spiroplasma species, lected in Georgia, USA. groups, and subgroups. Phylogenetically, closely related DNA G+C content (mol%): 26 ± 1 (Bd). to Spiroplasma diabroticae and various flower spiroplasmas Type strain: ATCC 51746, TABS-2. in trees constructed using 16S rRNA gene sequences. The Sequence accession no. (16S rRNA gene): AY189132. genome size of strain OBMG is 1270 kbp. Experimentally pathogenic for insects and embryonated chicken eggs. 16. Spiroplasma insolitum Hackett, Whitcomb, Tully, Rose, Source: isolated from flowers of tulip tree and magnolia Carle, Bové, Henegar, Clark, Clark, Konai, Adams and Wil- trees in Maryland, USA. Other strains have been collected liamson 1993, 276VP from coleopterous insects. in.so′li.tum. L. neut. adj. insolitum unusual or uncommon, DNA G+C content (mol%): 25 (T ). m to denote unusual base composition. Type strain: ATCC 29989, 23-6. Sequence accession no. (16S rRNA gene): AY189131. Cells in exponential phase are long, motile, helical cells that lack true cell walls and periplasmic fibrils. Colonies on 14. Spiroplasma gladiatoris Whitcomb, French, Tully, Gas- solid SP-4 medium containing 0.8 or 2.25% Noble agar are parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, diffuse, with small central zones of growth surrounded by VP Clark and Williamson 1997b, 718 small satellite colonies. Colonies on solid SP-4 medium con- gla.di.a¢to.ris. L. gen. n. gladiatoris of a gladiator, reflect- taining horse serum and 0.8% Noble agar show fried-egg ing the initial isolation of the organism from the horse fly morphology. Biological properties are listed in Table 142. Tabanus gladiator. Serologically distinct from other Spiroplasma species and Morphology is as described for the genus. Cells are groups, but cross-reacts reciprocally in complex patterns motile helical filaments. Colonies on solid medium contain- of relatedness with group I subgroups and Spiroplasma ing 3% Noble agar are granular with dense centers and dif- poulsonii. DNA–DNA renaturation experiments confirm fuse edges, do not have satellites, and never have a fried-egg that the differences between the type strain and other sub- appearance. Biological properties are listed in Table 142. groups of group I are great enough to warrant its designa- Serologically distinct from other Spiroplasma species, tion as a distinct species. Has close phylogenetic affinities groups, and subgroups. This species has a specific antigen, with other group I members and with Spiroplasma poulsonii Genus I. Spiroplasma 673

in trees ­constructed using 16S rRNA gene sequences. The members and with Spiroplasma poulsonii in trees constructed genome size is 1850 kbp (PFGE). Pathogenicity for insects using 16S rRNA gene sequences. The genome size is 1610 has not been determined. kbp (PFGE). Pathogenicity for plants and insects has been Source: the type strain was isolated from a fall flower (Aster- experimentally verified. aceae: Bidens sp.) collected in Maryland, USA. Similar iso- Source: isolated from maize displaying symptoms of corn lates have been found in the hemocoel of click beetles. Also stunt disease and from leafhoppers associated with diseased isolated from other composite and onagracead flowers and maize, largely in the neotropics.

from the guts of many insects visiting these flowers, includ- DNA G+C content (mol%): 26 ± 1 (Tm, Bd). ing cantharid and meloid beetles; syrphid flies; andrenid Type strain: ATCC 29320, E275. and megachilid bees; and four families of butterflies. Sequence accession no. (16S rRNA gene): DQ319068 (strain

DNA G+C content (mol%): 28 ± 1 (Tm, Bd). CR2-3x). Type strain: ATCC 33502, M55. 19. Spiroplasma lampyridicola Stevens, Tang, Jenkins, Goins, Sequence accession no. (16S rRNA gene): AY189133. Tully, Rose, Konai, Williamson, Carle, Bové, Hackett, 17. Spiroplasma ixodetis Tully, Rose, Yunker, Carle, Bové, Wil- French, Wedincamp, Henegar and Whitcomb 1997, 711VP VP liamson and Whitcomb 1995, 27 lam.py.ri.di¢co.la. N.L. n. Lampyridae the firefly beetle family; ix.o.de¢tis. N.L. gen. n. ixodetis of Ixodes, the genus name L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. of Ixodes pacificus ticks, from which the organism was first lampyridicola an inhabitant of members of the Lampyridae. isolated. The morphology is as described for the genus. Cells are Cells are coccoid forms, 300–500 nm in diameter, straight motile helical filaments. Colonies on solid medium contain- and branched filaments, or tightly coiled helical organisms. ing 3.0% Noble agar are small and granular with dense cen- Motility is flexional, but not translational. Colonies on solid ters, but do not have a true fried-egg appearance. Biological medium containing 2.25% Noble agar usually have the properties are listed in Table 142. appearance of fried eggs. Biological properties are listed in Serologically distinct from other Spiroplasma species, Table 142. groups, and subgroups. When tested as antigen, cross-reacts Serologically distinct from other Spiroplasma species, (one-way) with many specific spiroplasma antisera. Phylo- groups, and subgroups. Phylogenetically unique; occurs genetically, a sister to Spiroplasma leptinotarsae in trees con- at base of spiroplasma lineage in trees constructed using structed using 16S rRNA gene sequences. The genome size 16S rRNA gene sequences. The genome size is 2220 kbp is 1375 kbp (PFGE). (PFGE). Source: isolated from the gut fluids of a firefly beetle Source: isolated from macerated tissue suspensions pre- (Photuris pennsylvanicus) collected in Maryland, USA. Also pared from pooled adult Ixodes pacificus ticks (Ixodidae) known from Georgia and New Jersey, USA.

collected in Oregon, USA. DNA G+C content (mol%): 26 ± 1 (Tm, Bd).

DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 43206, PUP-1. Type strain: ATCC 33835, Y32. Sequence accession no. (16S rRNA gene): AY189134. Sequence accession no. (16S rRNA gene): M24477. 20. Spiroplasma leptinotarsae Hackett, Whitcomb, Clark, Hen- 18. Spiroplasma kunkelii Whitcomb, Chen, Williamson, Liao, egar, Lynn, Wagner, Tully, Gasparich, Rose, Carle, Bové, Tully, Bové, Mouches, Rose, Coan and Clark 1986, 175VP Konai, Clark, Adams and Williamson 1996b, 910VP kun.kel¢i.i. N.L. masc. gen. n. kunkelii of Kunkel, named lep.ti.no.tar¢sae. N.L. gen. n. leptinotarsae of Leptinotarsa, after Louis Otto Kunkel (1884–1960), to honor his major referring to Leptinotarsa decemlineata, the Colorado potato and fundamental contributions to the study of plant mol- beetle. licutes. Cells in vivo are usually seen in the resting stage, in which Cells in exponential phase are helical, motile filaments, they consist of coin-like compressed coils. When placed in 100–150 nm in diameter and 3–10 mm long to nonhelical fresh medium, these bodies turn immediately into “spring”- filaments or spherical cells, 300–800 nm in diameter. Colo- or “funnel”-shaped spirals, which are capable of very rapid nies on solid medium containing 0.8% Noble agar are usu- translational motility. After a relatively small number of ally diffuse, rarely exhibiting central zones of growth into passes in vitro, this spectacular morphology is lost and the agar. Colonies on solid C-3G medium containing 5% horse cells return to the modal morphology as described for the serum or on media containing 2.25% Noble agar frequently genus. Colonies on solid medium containing 2.0% Noble have a fried-egg morphology. Biological properties are agar are slightly diffuse to discrete and produce numerous listed in Table 142. satellites. Biological properties are listed in Table 142. Serologically distinct from other Spiroplasma species, Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares complex patterns of groups, and subgroups. When tested as antigen, cross-reacts reciprocal cross-reactivity with members of other group I with many spiroplasma antisera (one-way). Phylogeneti- subgroups and Spiroplasma poulsonii. DNA–DNA renatur- cally, a sister to Spiroplasma lampyridicola in trees constructed ation experiments confirm that the serological differences using 16S rRNA gene sequences. The genome size is 1,085 between the type strain and other subgroups of group I are kbp (PFGE). great enough to warrant its designation as a distinct spe- Source: isolated from the gut of Colorado potato beetle cies. Has close phylogenetic affinities with other group I (Leptinotarsa decemlineata) larvae in Maryland, USA. Also 674 Family II. Spiroplasmataceae

isolated from beetles collected in Maryland, Michigan, New two other tabanid spiroplasmas, Spiroplasma turonicum and Mexico, North Carolina, Texas, Canada, and Poland. Spiroplasma litorale, in trees constructed using 16S rRNA

DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC). gene sequences. The genome size is 1370 kbp (PFGE). Type strain: ATCC 43213, LD-1. Source: isolated from the gut of a female green-eyed horse Sequence accession no. (16S rRNA gene): AY189305. fly Tabanus( nigrovittatus) from the Outer Banks of North Carolina. Also collected from coastal Georgia and both 21. Spiroplasma leucomae Oduori, Lipa and Gasparich 2005, Atlantic and Pacific coasts of Costa Rica. 2449VP DNA G+C content (mol%): 25 ± 1 (Bd). leu.co¢mae. N.L. gen. n. leucomae of Leucoma, systematic Type strain: ATCC 34211, TN-1. genus name of the white satin moth (Lepidoptera: Lyman- Sequence accession no. (16S rRNA gene): AY189306. triidae), the source of the type strain. 24. Spiroplasma melliferum Clark, Whitcomb, Tully, Mouches, Morphology is as described for the genus. Cells are fila- Saillard, Bové, Wróblewski, Carle, Rose, Henegar and Wil- mentous, helical, motile, and approximately 150 nm in liamson 1985, 305VP diameter. They freely pass through filters with pores of 450 and 220 nm, but do not pass through filters with 100 nm mel.li′fe.rum. L. adj. mellifer, -fera, -ferum honey-bearing, pores. Biological properties are listed in Table 142. honey-producing; L. neut. adj. melliferum intended to mean Serologically distinct from previously established Spiro- isolated from the honey bee (Apis mellifera). plasma species, groups, and subgroups. The genome size Morphology is as described for the genus. Cells are pleo- has not been determined. Pathogenicity for the moth lar- morphic, varying from helical filaments that are 100–150 vae is not known. nm in diameter and 3–10 mm in length to nonhelical fila- Source: isolated from fifth instar satin moth larvae Leu( - ments or spherical cells that are 300–800 nm in diameter. coma salicis). The motile cells lack true cell wells and periplasmic fibrils.

DNA G+C content (mol%): 24 ± 1 (Tm). Colonies on solid medium supplemented with 0.8% Noble Type strain: ATCC BAA-521, NBRC 100392, SMA. agar are usually diffuse, rarely exhibiting central zones of Sequence accession no. (16S rRNA gene): DQ101278. growth into agar. Colonies on solid medium containing 2.25% Noble agar are smaller, but frequently have a fried- 22. Spiroplasma lineolae French, Whitcomb, Tully, Carle, Bové, egg morphology. Physiological and genomic properties are Henegar, Adams, Gasparich and Williamson 1997, 1080VP listed in Table 142. lin.e.o¢lae. N.L. n. lineola a species of tabanid fly; N.L. gen. Serologically distinct from other Spiroplasma species, n. lineolae of Tabanus lineola, from which the organism was groups, and subgroups, but shares complex patterns of isolated. reciprocal cross-reactivity with members of other group I The morphology is as described for the genus. Cells are subgroups and Spiroplasma poulsonii. Has close phyloge- motile, helical filaments, 200–300 nm in diameter. Colo- netic affinities with other group I members and with Spiro- nies on solid medium containing 3% Noble agar are small, plasma poulsonii in trees constructed using 16S rRNA gene granular, and never have a fried-egg appearance. Biological sequences. DNA–DNA renaturation experiments confirm properties are listed in Table 142. that the serological differences between the type strain and Serologically distinct from other Spiroplasma species, other subgroups of group I are great enough to warrant its groups, and subgroups. Phylogenetic position has not been designation as a distinct species. The genome size is 1460 determined, but its other taxonomic properties suggest that kbp (PFGE). Pathogenic for honey bees in natural and it may be related to other tabanid spiroplasmas of the Apis experimental oral infections. cluster. The genome size is 1390 kbp (PFGE). Source: isolated from hemolymph and gut of honey bees Source: type strain isolated from the viscera of the tabanid (Apis mellifera) in widely separated geographic regions. Also flyTabanus lineola collected in coastal Georgia. A strain from recovered from hemolymph of bumble bees, leafcutter Tabanus lineola has been collected in Costa Rica (Whitcomb bees, and a robber fly, and the intestinal contents of sweat et al., 2007). bees, digger bees, bumble bees, and a butterfly. Also recov-

DNA G+C content (mol%): 25 ± 1 (Tm, Bd). ered from a variety of plant surfaces (flowers) in widely Type strain: ATCC 51749, TALS-2. separated geographic regions.

Sequence accession no. (16S rRNA gene): DQ860100. DNA G+C content (mol%): 26–28 (Tm, Bd). Type strain: ATCC 33219, BC-3. 23. Spiroplasma litorale Konai, Whitcomb, French, Tully, Rose, Sequence accession no. (16S rRNA gene): AY325304. Carle, Bové, Hackett, Henegar, Clark and Williamson 1997, 361VP 25. Spiroplasma mirum Tully, Whitcomb, Rose and Bové 1982, VP li.to.ra¢le. L. neut. adj. litorale of the shore or coastal area. 99 The morphology is as described for the genus. Cells are mi′rum. L. neut. adj. mirum extraordinary. motile, helical filaments. Colonies on solid medium contain- The morphology is as described for the genus. Helical ing 2.25% Noble agar are granular with dense centers, uneven filaments measure 100–200 nm in diameter and 3–8 mm margins, and multiple satellites, and never have fried-egg in length. Colonies on solid media containing fetal bovine appearance. Biological properties are listed in Table 142. serum and 0.8–2.25% Noble agar (Difco) are diffuse and Serologically distinct from other Spiroplasma species, without central zones of growth into the agar. Solid media groups, and subgroups. Phylogenetically, closely related to prepared with 1.25% agar and in which fetal bovine serum Genus I. Spiroplasma 675

has been replaced with bovine serum fraction yield colonies Serologically distinct from other Spiroplasma species, with central zones of growth into the agar and no peripheral groups, and subgroups. Reacts reciprocally in deformation growth on the surface of the medium. Moderate turbidity is serology at very low levels in deformation tests with Spiro- produced during growth in liquid media. Biological proper- plasma apis. “Bridge strains” have been isolated in Georgia ties are listed in Table 142. This species has been cultivated with substantial cross-reactivity with both Spiroplasma mon- in a defined medium. tanense and Spiroplasma apis. Sister to Spiroplasma apis in trees Serologically distinct from other Spiroplasma species, constructed using 16S rRNA gene sequences. The genome groups, and subgroups. Phylogenetically, in trees con- size is 1225 kbp (PFGE). structed using 16S rRNA gene sequences, this species is Source: isolated from the gut of the tabanid fly Hybomitra basal to group I and group VIII spiroplasmas on the one opaca, in southwestern Montana. Other isolates have been hand, and to the Apis cluster and Entomoplasmataceae on the obtained from New England, Connecticut, and southeast- other. It is the most primitive (plesiomorphic) spiroplasma ern Canada. with modal helicity. The genome size is 1300 kbp (PFGE). DNA G+C content (mol%): 28 ± 1 (Bd). Produces experimental ocular and nervous system disease Type strain: ATCC 51745, HYOS-1. and death in intracerebrally inoculated suckling animals Sequence accession no. (16S rRNA gene): AY189307. (rats, mice, hamsters, and rabbits). Pathogenic for chicken 28. Spiroplasma penaei Nunan, Lightner, Oduori and Gas- embryos via yolk sac inoculation. Experimentally patho- parich 2005, 2320VP genic for the wax moth (Galleria mellonella). Source: the type strain was isolated from rabbit ticks (Hae- pe.na′e.i. N.L. n. Penaeus a species of shrimp; N.L. gen. maphysalis leporispalustris) collected in Georgia, USA. Other penaei of Penaeus, referring to Penaeus vannamei, from which strains have been collected in Georgia, Maryland, and New the organism was isolated. York, USA. The morphology is as described for the genus. Cells are heli-

DNA G+C content (mol%): 30–31 (Tm). cal and motile. Biological properties are listed in Table 142. Type strain: ATCC 29335, SMCA. Serologically distinct from previously characterized Spiro- Sequence accession no. (16S rRNA gene): M24662. plasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups. 26. Spiroplasma monobiae Whitcomb, Tully, Rose, Carle, Bové, Has close phylogenetic affinities with other group I mem- Henegar, Hackett, Clark, Konai, Adams and Williamson bers and with Spiroplasma poulsonii in trees constructed using 1993b, 259VP 16S rRNA gene sequences. The genome size has not been mo.no.bi′ae. N.L. n. Monobia a genus of vespid wasps; N.L. determined. Pathogenicity has been indicated by injection gen. n. monobiae of the genus Monobia, from which the into Penaeus vannamei. organism was isolated. Source: isolated from the hemolymph of the Pacific white The morphology is as described for the genus, with shrimp, Penaeus vannamei.

motile helical filaments. Colonies on solid medium contain- DNA G+C content (mol%): 29 ± 1 (Tm). ing 2.25% Noble agar are diffuse and never have a fried-egg Type strain: CAIM 1252, SHRIMP, ATCC BAA-1082. appearance. Biological properties are listed in Table 142. Sequence accession no. (16S rRNA gene): AY771927. Serologically distinct from other Spiroplasma species, 29. Spiroplasma phoeniceum Saillard, Vignault, Bové, Raie, groups, and subgroups. Phylogenetically, a member of the Tully, Williamson, Fos, Garnier, Gadeau, Carle and Whit- Apis clade, but with no especially close neighbors in trees comb 1987, 113VP constructed using 16S rRNA gene sequences. The genome size is 940 kbp (PFGE). phoe.ni¢ce.um. N.L. neut. adj. phoeniceum (from L. neut. Source: isolated from the hemolymph of an adult vespid adj. phonicium) of Phoenice, an ancient country that was wasp (Monobia quadridens) collected in Maryland, USA. located on today’s Syrian coast, referring to the geographi- Based on its residence in the gut of a flower-visiting insect, cal origin of the isolates. this species is thought to be transmitted on flowers. Morphology is as described for the genus. Colonies on

DNA G+C content (mol%): 28 ± 1 (Tm, Bd, HPLC). solid medium containing 0.8% Noble agar show fried-egg Type strain: ATCC 33825, MQ-1. morphology. Physiological and genomic properties are Sequence accession no. (16S rRNA gene): M24481. listed in Table 142. Serologically distinct from other Spiroplasma species, 27. Spiroplasma montanense Whitcomb, French, Tully, Rose, groups, and subgroups, but shares some cross-reactivity with Carle, Bové, Clark, Henegar, Konai, Hackett, Adams and members of other group I subgroups and Spiroplasma poul- Williamson 1997c, 722VP sonii. Has close phylogenetic affinities with other group I mon.ta.nen¢se. N.L. neut. adj. montanense pertaining to members and with Spiroplasma poulsonii in trees constructed Montana, where the species was first isolated. using 16S rRNA gene sequences. Has been shown to be The morphology is as described for the genus. Cells transmissible to leafhoppers by injection and experimen- are motile, helical filaments that lack a cell wall. Colo- tally pathogenic to aster inoculated by the injected leafhop- nies on solid medium containing 2.25% Noble agar are pers. DNA–DNA renaturation experiments confirm that the granular and have dense centers, irregular margins, and differences between the type strain and other subgroups of numerous small satellites. Biological properties are listed group I are great enough to warrant its designation as a dis- in Table 142. tinct species. The genome size is 1860 kbp (PFGE). 676 Family II. Spiroplasmataceae

Source: isolated from periwinkles that were naturally 32. Spiroplasma sabaudiense Abalain-Colloc, Chastel, Tully, infected in various locations along the Syrian coastal area. Bové, Whitcomb, Gilot and Williamson 1987, 264VP

DNA G+C content (mol%): 26 ± 1 (Tm, Bd). sa.bau.di.en¢se. L. neut. adj. sabaudiense of Sabaudia, an Type strain: ATCC 43115, P40. ancient country of Gaul, corresponding to present day Sequence accession no. (16S rRNA gene): AY772395. Savoy, referring to the geographic origin of the isolate. 30. Spiroplasma platyhelix Williamson, Adams, Whitcomb, Tul- The morphology is as described for the genus. Cells are ly, Carle, Konai, Bové and Henegar 1997, 766VP helical filaments, 100–160 nm in diameter and 3.1–3.8 mm pla.ty.he¢lix. Gr. adj. platys flat; Gr. fem. n. helix a coil or spi- long. Motile. Colonies on solid medium containing 1.6% ral; N.L. fem. n. platyhelix flat coil, referring to the flattened Noble agar are diffuse, rarely exhibiting fried-egg morphol- nature of the helical filament. ogy, with numerous satellite colonies. Physiological and genomic properties are listed in Table 142. Cells are flattened, helical filaments, 200–300 nm in Serologically distinct from other Spiroplasma species, diameter. They show no rotatory or translational motility, groups, and subgroups. Phylogenetically, related to Spiro- but exhibit contractile movements in which tightness of coil- plasma alleghenense and Spiroplasma sp. TIUS-1 in trees con- ing moves along the axis of the filament. Colonies on solid structed using 16S rRNA gene sequences. The genome size medium containing 2.25% Noble agar form perfect fried- is 1175 kbp (PFGE). egg colonies with dense centers, smooth edges, and without Source: isolated from a triturate of female Aedes spp. mos- satellites. Biological properties are listed in Table 142. quitoes in Savoy, France. Serologically distinct from other Spiroplasma spe- DNA G+C content (mol%): 30 ± 1 (T , Bd). cies, groups, and subgroups. The genome size is 780 kbp m Type strain: ATCC 43303, Ar-1343. (PFGE). Sequence accession no. (16S rRNA gene): AY189308. Source: isolated from the gut of a dragonfly, Pachydiplax longipennis, collected in Maryland, USA. 33. Spiroplasma syrphidicola Whitcomb, Gasparich, French, DNA G+C content (mol%): 29 ± 1 (Bd). Tully, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Type strain: ATCC 51748, PALS-1. Clark and Williamson 1996, 799VP Sequence accession no. (16S rRNA gene): AY800347. syr.phi.di¢co.la. N.L. pl. n. Syrphidae a family of flies; L. suff. 31. Spiroplasma poulsonii Williamson, Sakaguchi, Hackett, -cola (from L. masc. or fem. n. incola) inhabitant, dweller; Whitcomb, Tully, Carle, Bové, Adams, Konai and Henegar N.L. masc. n. syrphidicola inhabitant of syrphid flies, the 1999, 616VP insects from which the organism was isolated. poul.so′ni.i. N.L. masc. gen. n. poulsonii of Poulson, named Helical motile filaments are short and thin, passing a in memory of Donald F. Poulson, in whose laboratory at 220 nm filter quantitatively. Grows to titers as high as 1011/ Yale University this spiroplasma was discovered and studied ml. These short, thin, abundant cells are provisionally diag- intensively. nostic for group VIII. Colonies on solid medium contain- Morphology is as described for the genus. Long, motile, ing 2.25% Noble agar are irregular with satellites, diffuse, helical filaments, 200–250 nm in diameter occur in vivo and never have a fried-egg appearance. Growth on solid in Drosophila hemolymph and in vitro. Colonies on solid medium containing 1.6% Noble agar is diffuse. Biological medium containing 1.8% Noble agar are small (60–70 mm properties are listed in Table 142. in diameter), have dense centers and uneven edges, and Serologically distinct from other Spiroplasma species, are without satellites. Biological properties are listed in groups, and subgroups, but shares some reciprocal cross- Table 142. reactivity with members of other group VIII subgroups. Serologically distinct from other Spiroplasma species, Placement of group VIII strains into subgroups has become groups, and subgroups, but shares some reciprocal cross- increasingly difficult as more strains have accumulated. reactivity with members of group I subgroups. Phylogeneti- Phylogenetically, this species is closely related to other cally related to group I spiroplasmas in trees constructed group VIII strains in trees constructed using 16S rRNA using 16S rRNA gene sequences. The genome size is 2040 gene sequences. The 16S rRNA gene sequence similarity kbp (PFGE). Spiroplasmas causing sex-ratio abnormalities coefficients of group VIII spiroplasmas are >0.99, so this occur naturally in Drosophila spp. collected in Brazil, Colom- gene is insufficient for species separations in group VIII. bia, Dominican Republic, Haiti, Jamaica, and the West DNA–DNA renaturation experiments confirm that the dif- Indies. Non-male-lethal spiroplasmas also occur in natu- ferences between the type strain and other subgroups of ral populations of Drosophila hydei in Japan. Pathogenicity group VIII are great enough to warrant its designation as a (lethality to male progeny) has been confirmed by injection distinct species. Genome size is 1230 kbp (PFGE). into Drosophila pseudoobscura female flies. Vertical transmis- Source: isolated from the hemolymph of the syrphid fly sibility is lost after cultivation and cloning. Eristalis arbustorum in Maryland, USA. Strains that are pro- Source: isolated from the hemolymph of Drosophila pseu- visionally identified as Spiroplasma syrphidicola have been doobscura females infected by hemolymph transfer of the obtained from horse flies collected from several locations Barbados-3 strain of Drosophila willistoni SR organism. in the southeastern United States.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd). DNA G+C content (mol%): 30 ± 1 (Bd). Type strain: ATCC 43153, DW-1. Type strain: ATCC 33826, EA-1. Sequence accession no. (16S rRNA gene): M24483. Sequence accession no. (16S rRNA gene): AY189309. Genus I. Spiroplasma 677

34. Spiroplasma tabanidicola Whitcomb, French, Tully, Gas- 36. Spiroplasma turonicum Hélias, Vazeille-Falcoz, Le Goff, Ab- parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, alain-Colloc, Rodhain, Carle, Whitcomb, Williamson, Tully, Clark and Williamson 1997b, 718VP Bové and Chastel 1998, 460VP ta.ba.ni.di¢co.la. N.L. n. Tabanidae family name for horse tu.ro¢ni.cum. L. neut. adj. turonicum of Touraine, the flies; L. suff. -cola (from L. n. incola) inhabitant, dweller; province in France from which the organism was first iso- N.L. n. tabanidicola an inhabitant of horse flies. lated. The morphology is as described for the genus. Cells are The morphology is as described for the genus. Cells are motile, helical filaments that lack a cell wall. Colonies on motile, helical filaments. Colonies on solid medium con- solid medium containing 3% Noble agar are uneven and taining 3% Noble agar exhibit a “cauliflower-like” appear- granular with dense centers and irregular edges, do not ance and do not have a fried-egg morphology. Biological have satellites, and never have a fried-egg appearance. Phys- properties are listed in Table 142. iological and genomic properties are listed in Table 142. Serologically distinct from previously established Spiro- Serologically distinct from other Spiroplasma species, plasma species. Phylogenetically, related to two other tabanid groups, and subgroups. However, some strains may show spiroplasmas, Spiroplasma corruscae and Spiroplasma litorale, a very low level reciprocal serological cross-reaction in in trees constructed using 16S rRNA gene sequences. The deformation serology with Spiroplasma gladiatoris. This spe- genome size is 1305 kbp (PFGE). cies has a specific antigen, common to several spiroplasmal Source: isolated from a triturate of a single horse fly Hae( - inhabitants of horse flies, that confers a high level one-way matopota pluvialis) collected in France. cross-reaction when it is used as antigen. The genome size DNA G+C content (mol%): 25 ± 1 (Bd). is 1375 kbp (PFGE). Type strain: ATCC 700271, Tab4c. Source: isolated from the gut of a horse fly belonging to Sequence accession no. (16S rRNA gene): AY189310. the Tabanus abdominalis-limbatinevris complex. 37. Spiroplasma velocicrescens Konai, Whitcomb, Tully, Rose, DNA G+C content (mol%): 26 ± 1 (Bd). Carle, Bové, Henegar, Hackett, Clark and Williamson 1995, Type strain: ATCC 51747, TAUS-1. 205VP Sequence accession no. (16S rRNA gene): DQ004931. ve.lo.ci.cres¢cens. L. adj. velox, -ocis fast, quick; L. part. 35. Spiroplasma taiwanense Abalain-Colloc, Rosen, Tully, Bové, adj. crescens growing; N.L. n. part. adj. velocicrescens fast- VP Chastel and Williamson 1988, 105 growing. tai.wan.en¢se. N.L. neut. adj. taiwanense of or belonging to The morphology is as described for the genus. Cells are Taiwan, referring to the geographic origin of the isolate. helical, motile filaments, 200–300 nm in diameter. Colonies The morphology is as described for the genus. Cells are on solid medium containing 0.8% Noble agar are diffuse motile, helical filaments, 100–160 nm in diameter and 3.1– and never have a fried-egg appearance. Biological proper- 3.8 mm long. Colonies on solid medium containing 1.6% ties are listed in Table 142. Noble agar have fried-egg morphology. Biological proper- Serologically distinct from other Spiroplasma species, ties are listed in Table 142. groups, and subgroups. Phylogenetically, this species is Serologically distinct from other Spiroplasma species, sister to Spiroplasma chinense in trees constructed using groups, and subgroups. Phylogenetically, this species is in the 16S rRNA gene sequences. The genome size is 1480 kbp classical Apis cluster of spiroplasmas, but does not have an (PFGE). especially close neighbor in trees constructed using 16S rRNA Source: isolated from the gut of a vespid wasp, Monobia gene sequences. The genome size is 1195 kbp (PFGE). quadridens, collected in Maryland, USA. Based on its resi- Source: isolated from a triturate of female mosquitoes (Culex dence in the gut of a flower-visiting insect, this species is tritaeniorhynchus) at Taishan, Taiwan, Republic of China. thought to be transmitted on flowers.

DNA G+C content (mol%): 25 ± 1 (Tm, Bd). DNA G+C content (mol%): 27 ± 1 (Tm, Bd, HPLC). Type strain: ATCC 43302, CT-1. Type strain: ATCC 35262, MQ-4. Sequence accession no. (16S rRNA gene): M24476. Sequence accession no. (16S rRNA gene): AY189311.

References Adams, J.R., R.F. Whitcomb, J.G. Tully, E.A. Clark, D.L. Rose, P. Carle, M. Konai, J.M. Bové, R.B. Henegar and D.L. Williamson. 1997. Abalain-Colloc, M.L., C. Chastel, J.G. Tully, J.M. Bové, R.F. Whitcomb, Spiroplasma alleghenense sp. nov., a new species from the scorpion fly B. Gilot and D.L. Williamson. 1987. Spiroplasma sabaudiense sp. nov. Panorpa helena (Mecoptera: Panorpidae). Int. J. Syst. Bacteriol. 47: from mosquitos collected in France. Int. J. Syst. Bacteriol. 37: 260– 759–762. 265. Alexeeva, I., E.J. Elliott, S. Rollins, G.E. Gasparich, J. Lazar and R.G. Abalain-Colloc, M.L., L. Rosen, J.G. Tully, J.M. Bové, C. Chastel and Rohwer. 2006. Absence of Spiroplasma or other bacterial 16S rRNA D.L. Williamson. 1988. Spiroplasma taiwanense sp. nov. from Culex tri- genes in brain tissue of hamsters with scrapie. J. Clin. Microbiol. 44: taeniorhynchus mosquitos collected in Taiwan. Int. J. Syst. Bacteriol. 91–97. 38: 103–107. Alivizatos, A.S. 1988. Isolation and culture of corn stunt Spiroplasma in Abalain-Colloc, M.L., D.L. Williamson, P. Carle, J.H. Abalain, F. Bon- serum-free medium. J. Phytopathol. 122: 68–75. net, J.G. Tully, M. Konai, R.F. Whitcomb, J.M. Bové and C. Chastel. Aluotto, B.B., R. G. Wittler, C.O.Williams and J. E. Faber. 1970. Stan- 1993. Division of group XVI spiroplasmas into subgroups. Int. J. Syst. dardized bacteriologic techniques for characterization of Mycoplasma Bacteriol. 43: 342–346. species. Int. J. Syst. Bacteriol. 20: 35–58. 678 Family II. Spiroplasmataceae

Amikam, D., S. Razin and G. Glaser. 1982. Ribosomal RNA genes in Bévén, L. and H. Wróblewski. 1997. Effect of natural amphipathic pep- Mycoplasma. Nucleic Acids Res. 10: 4215–4222. tides on viability, membrane potential, cell shape and motility of mol- Amikam, D., G. Glaser and S. Razin. 1984. Mycoplasmas (Mollicutes) licutes. Res. Microbiol. 148: 163–175. have a low number of rRNA genes. J. Bacteriol. 158: 376–378. Bévén, L., D. Duval, S. Rebuffat, F.G. Riddell, B. Bodo and H. Wró- Ammar, E. and S.A. Hogenhout. 2005. Use of immunofluorescence con- blewski. 1998. Membrane permeabilisation and antimycoplasmic focal laser scanning microscopy to study distribution of the bacterium activity of the 18-residue peptaibols, trichorzins PA. Biochim. Bio- corn stunt spiroplasma in vector leafhoppers (Hemiptera: Cicadelli- phys. Acta 1372: 78–90. dae) and in host plants. Ann. Entomol. Soc. Am. 98: 820–826. Bi, K., H. Huang, W. Gu, J. Wang and W. Wang. 2008. Phylogenetic Ammar, E.D., D. Fulton, X. Bai, T. Meulia and S.A. Hogenhout. 2004. An analysis of Spiroplasmas from three freshwater crustaceans (Eriocheir attachment tip and pili-like structures in insect- and plant-pathogenic sinensis, Procambarus clarkia and Penaeus vannamei) in China. J. Inver- spiroplasmas of the class Mollicutes. Arch. Microbiol. 181: 97–105. tebr. Pathol. 99: 57–65. Anbutsu, H. and T. Fukatsu. 2003. Population dynamics of male-killing Boutareaud, A., J.L. Danet, M. Garnier and C. Saillard. 2004. Disruption and non-male-killing spiroplasmas in Drosophila melanogaster. Appl. of a gene predicted to encode a solute binding protein of an ABC Environ. Microbiol. 69: 1428–1434. transporter reduces transmission of Spiroplasma citri by the leafhop- André, A., W. Maccheroni, F. Doignon, M. Garnier and J. Renaudin. 2003. per Circulifer haematoceps. Appl. Environ. Microbiol. 70: 3960–3967. Glucose and trehalose PTS permeases of Spiroplasma citri probably share Bové, J.M. and C. Saillard. 1979. Cell biology of spiroplasmas. In The a single IIA domain, enabling the spiroplasma to adapt quickly to car- Mycoplasmas, vol. 3 (edited by Whitcomb and Tully). Academic bohydrate changes in its environment. Microbiology 149: 2687–2696. Press, New York, pp. 83–153. André, A., M. Maucourt, A. Moing, D. Rolin and J. Renaudin. 2005. Bové, J.M., C. Saillard, P. Junca, J.R. DeGorce-Dumas, B. Ricard, A. Sugar import and phytopathogenicity of Spiroplasma citri: glucose and Nhami, R.F. Whitcomb, D. Williamson and J.G. Tully. 1982. Guanine- fructose play distinct roles. Mol. Plant. Microbe. Interact. 18: 33–42. plus-cytosine content, hybridization percentages, and EcoRI restric- Archer, D.B., J. Best and C. Barber. 1981. Isolation and restriction map- tion enzyme profiles of spiroplasmal DNA. Rev. Infect. Dis. 4 Suppl: ping of a spiroplasma plasmid. J. Gen. Microbiol. 126: 511–514. S129–136. Bai, X. and S.A. Hogenhout. 2002. A genome sequence survey of the Bové, J.M., C. Mouches, P. Carle-Junca, J.R. Degorce-Dumas, J.G. Tully mollicute corn stunt spiroplasma Spiroplasma kunkelii. FEMS Micro- and R.F. Whitcomb. 1983. Spiroplasmas of Group I: the Spiroplasma biol. Lett. 210: 7–17. citri cluster. Yale J. Biol. Med. 56: 573–582. Barile, M.F. 1983. Arginine hydrolysis. In Methods in Mycoplasmology, vol. Bové, J.M., P. Carle, M. Garnier, F. Laigret, J. Renaudin and C. Saillard. 1 (edited by Razin and Tully). Academic Press, New York, pp. 345–349. 1989. Molecular and cellular biology of spiroplasmas. In The Myco- Baseman, J.B. and J.G. Tully. 1997. Mycoplasmas: sophisticated, re-emerg- plasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, ing, and burdened by their notoriety. Emerg. Infect. Dis. 3: 21–32. New York, pp. 243–364. Bastian, F.O. 1979. Spiroplasma-like inclusions in Creutzfeldt-Jakob dis- Bové, J.M. 1993. Molecular features of mollicutes. Clin. Infect. Dis. 17 ease. Arch. Pathol. Lab. Med. 103: 665–669. Suppl 1: S10–31. Bastian, F.O. 2005. Spiroplasma as a candidate agent for the transmis- Bové, J.M., X. Foissac and C. Saillard. 1993. Spiralins. In Subcellular sible spongiform encephalopathies. J. Neuropathol. Exp. Neurol. 64: Biochemistry. Mycoplasma Cell Membranes (edited by Rottem and 833–838. Kahane). Plenum Press, New York, pp. 203–223. Bastian, F.O. and J.W. Foster. 2001. Spiroplasma sp. 16S rDNA in Bové, J.M. 1997. Spiroplasmas: infectious agents of plants, arthropods Creutzfeldt-Jakob disease and scrapie as shown by PCR and DNA and vertebrates. Wien. Klin. Wochenschr. 109: 604–612. sequence analysis. J. Neuropathol. Exp. Neurol. 60: 613–620. Bové, J.M., J. Renaudin, C. Saillard, X. Foissac and M. Garnier. 2003. Bastian, F.O., S. Dash and R.F. Garry. 2004. Linking chronic wasting dis- Spiroplasma citri, a plant pathogenic mollicute: relationships with its ease to scrapie by comparison of Spiroplasma mirum ribosomal DNA two hosts, the plant and the leafhopper vector. Annu. Rev. Phyto- sequences. Exp. Mol. Pathol. 77: 49–56. pathol. 41: 483–500. Bastian, F.O. and C.D. Fermin. 2005. Slow virus disease: deciphering Bowyer, J.W. and E.C. Calavan. 1974. Antibiotic sensitivity in vitro of the conflicting data on the transmissible spongiform encephalopathies mycoplasmalike organism associated with citrus stubborn disease. (TSE) also called prion diseases. Microsc. Res. Tech. 68: 239–246. Phytopathology 64: 346–349. Bastian, F.O., D.E. Sanders, W.A. Forbes, S.D. Hagius, J.V. Walker, W.G. Brenner, C., H. Duclohier, V. Krchnak and H. Wroblewski. 1995. Confor- Henk, F.M. Enright and P.H. Elzer. 2007. Spiroplasma spp. from trans- mation, pore-forming activity, and antigenicity of synthetic peptide missible spongiform encephalopathy brains or ticks induce spongiform analogues of a spiralin putative amphipathic alpha helix. Biochim. encephalopathy in ruminants. J. Med. Microbiol. 56: 1235–1242. Biophys. Acta 1235: 161–168. Bébéar, C.M., P. Aullo, J.M. Bove and J. Renaudin. 1996. Spiroplasma citri Breton, M., S. Duret, N. Arricau-Bouvery, L. Beven and J. Renaudin. virus SpV1: characterization of viral sequences present in the spiro- 2008a. Characterizing the replication and stability regions of Spiro- plasma host chromosome. Curr. Microbiol. 32: 134–140. plasma citri plasmids identifies a novel replication protein and Bentley, J.K., Z. Veneti, J. Heraty and G.D. Hurst. 2007. The pathology expands the genetic toolbox for plant-pathogenic spiroplasmas. of embryo death caused by the male-killing Spiroplasma bacterium in Microbiology 154: 3232–3244. Drosophila nebulosa. BMC Biol. 5: 9. Breton, M., S. Duret, J.L. Danet, M.P. Dubrana and J. Renaudin. 2010. Berg, M., U. Melcher and J. Fletcher. 2001. Characterization of Spiro- Sequences essential for transmission of Spiroplasma citri leafhopper vec- plasma citri adhesion related protein SARP1, which contains a domain tor, Circulifer haematoceps, revealed by plasmid curing and replacement of a novel family designated sarpin. Gene 275: 57–64. based on incompatibility. Appl. Environ. Microbiol. 76: 3198–3205. Berho, N., S. Duret, J.L. Danet and J. Renaudin. 2006a. Plasmid pSci6 Brown, D.R., R.F. Whitcomb and J.M. Bradbury. 2007. Revised minimal from Spiroplasma citri GII-3 confers insect transmissibility to the non- standards for description of new species of the class Mollicutes (divi- transmissible strain S. citri 44. Microbiology 152: 2703–2716. sion Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. Berho, N., S. Duret and J. Renaudin. 2006b. Absence of plasmids encod- Calavan, E.C. and J.M. Bové. 1989. Molecular and cellular biology of ing adhesion-related proteins in non-insect-transmissible strains of spiroplasmas. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Spiroplasma citri. Microbiology 152: 873–886. Tully). Academic Press, New York, pp. 425–485. Bévén, L., M. LeHenaff, C. Fontenelle and H. Wróblewski. 1996. Inhibi- Carle, P., J.G. Tully, R.F. Whitcomb and J.M. Bové. 1990. Size of the tion of spiralin processing by the lipopeptide antibiotic globomycin. spiroplasmal genome and guanosine plus cytosine content of spiro- Curr. Microbiol. 33: 317–322. plasmal DNA. Zentralbl. Bakteriol. Suppl. 20: 926–931. Genus I. Spiroplasma 679

Carle, P., F. Laigret, J.G. Tully and J.M. Bové. 1995. Heterogeneity of Clark, T.B. 1984. Diversity of spiroplasma host-parasite relationships. genome sizes within the genus Spiroplasma. Int. J. Syst. Bacteriol. 45: Isr. J. Med. Sci. 20: 995–997. 178–181. Clark, T.B., R.F. Whitcomb, J.G. Tully, C. Mouches, C. Saillard, J.M. Carle, P., C. Saillard, N. Carrere, S. Carrere, S. Duret, S. Eveillard, P. Bové, H. Wroblewski, P. Carle, D.L. Rose, R.B. Henegar and D.L. Wil- Gaurivaud, G. Gourgues, J. Gouzy, P. Salar, E. Verdin, M. Breton, A. liamson. 1985. Spiroplasma melliferum, a new species from the honey- Blanchard, F. Laigret, J.M. Bové, J. Renaudin and X. Foissac. 2010. bee (Apis mellifera). Int. J. Syst. Bacteriol. 35: 296–308. Partial chromosome sequence of Spiroplasma citri reveals extensive Cohen, A.J., D.L. Williamson and K. Oishi. 1987. SpV3 viruses of Droso- viral invasion and important gene decay. Appl. Environ. Microbiol. phila spiroplasmas. Isr. J. Med. Sci. 23: 429–433. 76: 3420–3446. Cohen, A.J. and D.L. Williamson. 1988. Yeast supported growth of Droso- Carle, P., R.F. Whitcomb, K.J. Hackett, J.G. Tully, D.L. Rose, J.M. Bové, phila species spiroplasmas. Proceedings of the 7th International Con- R.B. Henegar, M. Konai and D.L. Williamson. 1997. Spiroplasma dia- gress of the International Organization for Mycoplasmology, Vienna, broticae sp. nov., from the southern corn rootworm beetle, Diabrotica Austria. undecimpunctata (Coleoptera: Chrysomelidae). Int. J. Syst. Bacteriol. Cohen, A.J., D.L. Williamson and P.R. Brink. 1989. A motility mutant 47: 78–80. of Spiroplasma melliferum induced with nitrous acid. Curr. Microbiol. Chang, C.J. and T.A. Chen. 1982. Spiroplasmas: cultivation in chemically 18: 219–222. defined medium. Science 215: 1121–1122. Cole, R.M., J.G. Tully, T.J. Popkin and J.M. Bové. 1973. Morphology, Chang, C.J. 1989. Nutrition and cultivation of spiroplasmas. In The ultrastructure, and bacteriophage infection of the helical myco- Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic plasma-like organism (Spiroplasma citri gen. nov., sp. nov.) cultured Press, New York, pp. 201–241. from “stubborn” disease of citrus. J. Bacteriol. 115: 367–384. Charbonneau, D.L. and W.C. Ghiorse. 1984. Ultrastructure and loca- Cole, R.M., J.G. Tully and T.J. Popkin. 1974. Virus-like particles in Spiro- tion of cytoplasmic fibrils in Spiroplasma-Floricola OBMG. Curr. Micro- plasma citri. Colloq. Inst. Natl. Santé Rech. Med. 33: 125–132. biol. 10: 65–71. Cole, R.M., W.O. Mitchell and C.F. Garon. 1977. Spiroplasma citri 3: Charron, A., C. Bébéar, G. Brun, P. Yot, J. Latrille and J.M. Bové. 1979. propagation, purification, proteins, and nucleic acid. Science 198: Separation and partial characterization of two deoxyribonucleic acid 1262–1263. polymerases from Spiroplasma citri. J. Bacteriol. 140: 763–768. Cole, R.M. 1979. Mycoplasma and Spiroplasma viruses: ultrastructure. Charron, A., M. Castroviejo, C. Bébéar, J. Latrille and J.M. Bové. 1982. A In The Mycoplasmas, vol. 1 (edited by Barile and Razin). Academic third DNA polymerase from Spiroplasma citri and two other spiroplas- Press, New York, pp. 385–410. mas. J. Bacteriol. 149: 1138–1141. Dally, E.L., T.S. Barros, Y. Zhao, S. Lin, B.A. Roe and R.E. Davis. 2006. Chastel, C., B. Gilot, F. Le Goff, B. Divau, G. Kerdraon, I. Humphery- Physical and genetic map of the Spiroplasma kunkelii CR2–3x chromo- Smith, R. Gruffax and A.M. Simitzis-Le Flohic. 1990. New develop- some. Can. J. Microbiol. 52: 857–867. ments in the ecology of mosquito spiroplasmas. Zentralbl. Bakteriol. Daniels, M.J., J.M. Longland and J. Gilbart. 1980. Aspects of motility Suppl. 20: 445–460. and chemotaxis in spiroplasmas. J. Gen. Microbiol. 118: 429–436. Chastel, C. and I. Humphery-Smith. 1991. Mosquito spiroplasmas. Adv. Daniels, M.J. and J.M. Longland. 1984. Chemotactic behavior of spiro- Dis. Vector Res. 7: 149–205. plasmas. Curr. Microbiol. 10: 191–193. Chastel, C., F. Le Goff and I. Humphery-Smith. 1991. Multiplication Davis, R.E., J.F. Worley, R.F. Whitcomb, T. Ishijima and R.L. Steere. and persistence of Spiroplasma melliferum strain A56 in experimentally 1972a. Helical filaments produced by a mycoplasma-like organism infected suckling mice. Res. Microbiol. 142: 411–417. associated with corn stunt disease. Science 176: 521–523. Chen, T.A. and C.H. Liao. 1975. Corn stunt spiroplasma: Isolation, cul- Davis, R.E., R.F. Whitcomb, T.A. Chen and R.R. Granados. 1972b. tivation and proof of pathogenesis. Science 188: 1015–1017. Current status of the aetiology of corn stunt disease. In Pathogenic Chevalier, C., C. Saillard and J.M. Bové. 1990. Organization and nucle- Mycoplasmas (edited by Elliott and Birch). Elsevier-Excerpta Med- otide sequences of the Spiroplasma citri genes for ribosomal protein ica-North-Holland, Amsterdam, pp. 205–214. S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate Davis, R.E. and J.F. Worley. 1973. Spiroplasma: Motile, helical microorgan- kinase, and an unidentified protein. J. Bacteriol. 172: 2693–2703. ism associated with corn stunt disease. Phytopathology 63: 403–408. Chipman, P.R., M. Agbandje-McKenna, J. Renaudin, T.S. Baker and R. Davis, R.E. 1978. Spiroplasma associated with flowers of tulip tree (Liri- McKenna. 1998. Structural analysis of the Spiroplasma virus, SpV4: odendron tulipifera L). Can. J. Microbiol. 24: 954–959. implications for evolutionary variation to obtain host diversity among Davis, R.E., I.M. Lee and J.F. Worley. 1981. Spiroplasma floricola, a new the Microviridae. Structure 6: 135–145. species isolated from surfaces of flowers of the tulip tree, Liriodendron Christiansen, C., G. Askaa, E.A. Freundt and R.F. Whitcomb. 1979. tulipifera L. Int. J. Syst. Bacteriol. 31: 456–464. Nucleic-acid hybridization experiments with Spiroplasma citri and the Davis, R.E., E.L. Dally, R. Jomantiene, Y. Zhao, B. Roe, S. Lin and J. Shao. corn stunt and suckling mouse cataract spiroplasmas. Curr. Micro- 2005. Cryptic plasmid pSKU146 from the wall-less plant pathogen biol. 2: 323–326. Spiroplasma kunkelii encodes an adhesin and components of a type IV Citti, C., L. Marechal-Drouard, C. Saillard, J.H. Weil and J.M. Bové. translocation-related conjugation system. Plasmid 53: 179–190. 1992. Spiroplasma citri UGG and UGA tryptophan codons: sequence DeSoete, G. 1983. A least square algorithm for fitting additive trees to of the two tryptophanyl-tRNAs and organization of the correspond- proximity data. Psychometrika 48: 621–626. ing genes. J. Bacteriol. 174: 6471–6478. Dickinson, M.J. and R. Townsend. 1984. Characterization of the Clark, HF. 1964. Suckling mouse cataract agent. J. Infect. Dis. 114: genome of a rod-shaped virus Infecting Spiroplasma citri. J. Gen. Virol. 476–487. 65: 1607–1610. Clark, HF. and L.B. Rorke. 1979. Spiroplasmas of tick origin and their Duret, S., J.L. Danet, M. Garnier and J. Renaudin. 1999. Gene dis- pathogenicity. In The Mycoplasmas, vol. 3 (edited by Whitcomb and ruption through homologous recombination in Spiroplasma citri: Tully). Academic Press, New York, pp. 155–174. an scm1-disrupted motility mutant is pathogenic. J. Bacteriol. 181: Clark, T.B. 1977. Spiroplasma sp., a new pathogen in honey bees. J. Inver- 7449–7456. tebr. Pathol. 29: 112–113. Duret, S., N. Berho, J.L. Danet, M. Garnier and J. Renaudin. 2003. Clark, T.B. 1978. Honey bee spiroplasmosis, a new problem for bee- Spiralin is not essential for helicity, motility, or pathogenicity but keepers. Am. Bee J. 118: 18–19. is required for efficient transmission of Spiroplasma citri by its leaf- Clark, T.B. 1982. Spiroplasmas: diversity of arthropod reservoirs and hopper vector Circulifer haematoceps. Appl. Environ. Microbiol. 69: host-parasite relationships. Science 217: 57–59. 6225–6234. 680 Family II. Spiroplasmataceae

Duret, S., A. Andre and J. Renaudin. 2005. Specific gene targeting Gasparich, G.E., K.J. Hackett, E.A. Clark, J. Renaudin and R.F. Whit- in Spiroplasma citri: improved vectors and production of unmarked comb. 1993a. Occurrence of extrachromosomal deoxyribonucleic mutations using site-specific recombination. Microbiology 151: acids in spiroplasmas associated with plants, insects, and ticks. Plas- 2793–2803. mid 29: 81–93. Ebbert, M. and L.R. Nault. 1994. Improved overwintering ability in Gasparich, G.E., K.J. Hackett, C. Stamburski, J. Renaudin and J.M. Dalbulus maidis (Homoptera: Cicadellidae) vectors infected with Bové. 1993b. Optimization of methods for transfecting Spiroplasma Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae). Environ. citri strain R8A2 HP with the spiroplasma virus SpV1 replicative form. Entomol. 23: 634–644. Plasmid 29: 193–205. Enigl, M. and P. Schausberger. 2007. Incidence of the endosymbionts Gasparich, G.E., C. Saillard, E.A. Clark, M. Konai, F.E. French, J.G. Wolbachia, Cardinium and Spiroplasma in phytoseiid mites and associ- Tully, K.J. Hackett and R.F. Whitcomb. 1993c. Serologic and genomic ated prey. Exp. Appl. Acarol. 42: 75–85. relatedness of group-VIII and group-XVII spiroplasmas and subdivi- FAO/WHO. 1974. Preservation of mycoplasmas by lyophilization. sion of spiroplasma group-VIII into subgroups. Int. J. Syst. Bacteriol. World Health Organization working document VPH/MIC/741. 43: 338–341. FAO/WHO Programme on Comparative Mycoplasmology Working Gasparich, G.E. and K.J. Hackett. 1994. Characterization of a cryptic Group. World Health Organization, Geneva. extrachromosomal element isolated from the mollicute Spiroplasma Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) 3.57 edn. taiwanense. Plasmid 32: 342–343. Department of Genetics, University of Washington, Seattle. Gasparich, G.E., K.J. Hackett, F.E. French and R.F. Whitcomb. 1998. Fletcher, J., A. Wayadande, U. Melcher and F.C. Ye. 1998. The phyto- Serologic and genomic relatedness of group XIV spiroplasma iso- pathogenic mollicute–insect vector interface: a closer look. Phytopa- lates from a lampyrid beetle and tabanid flies: an ecologic paradox. thology 88: 1351–1358. Int. J. Syst. Bacteriol. 48: 321–324. Foissac, X., C. Saillard, J. Gandar, L. Zreik and J.M. Bové. 1996. Spiralin Gasparich, G.E., R.F. Whitcomb, D. Dodge, F.E. French, J. Glass and polymorphism in strains of Spiroplasma citri is not due to differences D.L. Williamson. 2004. The genus Spiroplasma and its non-helical in posttranslational palmitoylation. J. Bacteriol. 178: 2934–2940. descendants: phylogenetic classification, correlation with phenotype Foissac, X., J.M. Bové and C. Saillard. 1997a. Sequence analysis of Spiro- and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Micro- plasma phoeniceum and Spiroplasma kunkelii spiralin genes and com- biol. 54: 893–918. parison with other spiralin genes. Curr. Microbiol. 35: 240–243. Gaurivaud, P., F. Laigret and J.M. Bové. 1996. Insusceptibility of mem- Foissac, X., J.L. Danet, C. Saillard, P. Gaurivaud, F. Laigret, C. Paré bers of the class Mollicutes to rifampin: studies of the Spiroplasma citri and J.M. Bové. 1997b. Mutagenesis by insertion of Tn4001 into the RNA polymerase b-subunit gene. Antimicrob. Agents Chemother. 40: genome of Spiroplasma citri: Characterization of mutants affected in 858–862. plant pathogenicity and transmission to the plant by the leafhop- Gaurivaud, P., J.L. Danet, F. Laigret, M. Garnier and J.M. Bové. 2000a. per vector Circulifer haematoceps. Mol. Plant Microbe Interact. 10: Fructose utilization and phytopathogenicity of Spiroplasma citri. Mol. 454–461. Plant Microbe Interact. 13: 1145–1155. Foissac, X., C. Saillard and J.M. Bové. 1997c. Random insertion of trans- Gaurivaud, P., F. Laigret, M. Garnier and J.M. Bové. 2000b. Fructose poson Tn4001 in the genome of Spiroplasma citri strain GII3. Plasmid utilization and pathogenicity of Spiroplasma citri: characterization of 37: 80–86. the fructose operon. Gene 252: 61–69. French, F.E., R.F. Whitcomb, J.G. Tully, K.J. Hackett, E.A. Clark, R.B. Gaurivaud, P., F. Laigret, E. Verdin, M. Garnier and J.M. Bové. 2000c. Henegar, A.G. Wagner and D.L. Rose. 1990. Tabanid spiroplasmas of Fructose operon mutants of Spiroplasma citri. Microbiology 146: the southeast USA: new groups and correlation with host life history 2229–2236. strategy. Zentralbl. Bakteriol. Suppl. 20: 919–922. Gaurivaud, P., F. Laigret, M. Garnier and J.M. Bove. 2001. Characteriza- French, F.E., R.F. Whitcomb, J.G. Tully, D.L. Williamson and R.B. Hen- tion of FruR as a putative activator of the fructose operon of Spiro- egar. 1996. Spiroplasmas of Tabanus lineola. IOM Lett. 4: 211–212. plasma citri. FEMS Microbiol. Lett. 198: 73–78. French, F.E., R.F. Whitcomb, J.G. Tully, P. Carle, J.M. Bové, R.B. Hen- Gilad, R., A. Porat and S. Trachtenberg. 2003. Motility modes of Spiro- egar, J.R. Adams, G.E. Gasparich and D.L. Williamson. 1997. Spiro- plasma melliferum BC3: a helical, wall-less bacterium driven by a linear plasma lineolae sp. nov., from the horsefly Tabanus lineola (Diptera: motor. Mol. Microbiol. 47: 657–669. Tabanidae). Int. J. Syst. Bacteriol. 47: 1078–1081. Goodacre, S.L., O.Y. Martin, C.F. Thomas and G.M. Hewitt. 2006. Wol- Fukatsu, T. and N. Nikoh. 1998. Two intracellular symbiotic bacteria bachia and other endosymbiont infections in spiders. Mol. Ecol. 15: from the mulberry psyllid Anomoneura mori (Insecta, Homoptera). 517–527. Appl. Environ. Microbiol. 64: 3599–3606. Gordon, D.T., L.R. Nault, N.H. Gordon and S.E. Heady. 1985. Sero- Fukatsu, T. and N. Nikoh. 2000. Endosymbiotic microbiota of the bam- logical detection of corn stunt spiroplasma and maize rayado fino boo pseudococcid Antonina crawii (Insecta, Homoptera). Appl. Envi- virus in field-collected Dalbulus spp. from Mexico. Plant Dis. 69: ron. Microbiol. 66: 643–650. 108–111. Fukatsu, T., T. Tsuchida, N. Nikoh and R. Koga. 2001. Spiroplasma sym- Grau, O., F. Laigret and J.M. Bové. 1988. Analysis of ribosomal RNA biont of the pea aphid, Acyrthosiphon pisum (Insecta: Homoptera). genes in two spiroplasmas, one acholeplasma and one unclassified Appl. Environ. Microbiol. 67: 1284–1291. mollicute. Zentralbl. Bakteriol. Suppl. 20: 895–897. Gadeau, A.P., C. Mouches and J.M. Bové. 1986. Probable insensitivity Grulet, O., I. Humphery-Smith, C. Sunyach, F. Le Goff and C. Chastel. of mollicutes to rifampin and characterization of spiroplasmal DNA- 1993. Spiromed: a rapid and inexpensive spiroplasma isolation tech- dependent RNA polymerase. J. Bacteriol. 166: 824–828. nique. J. Microbiol. Methods 17: 123–128. Garnier, M., M. Clerc and J.M. Bové. 1981. Growth and division of Guo, Y.H., T.A. Chen, R.F. Whitcomb, D.L. Rose, J.G. Tully, D.L. Wil- spiroplasmas: morphology of Spiroplasma citri during growth in liquid liamson, X.D. Ye and Y.X. Chen. 1990. Spiroplasma chinense sp. nov. medium. J. Bacteriol. 147: 642–652. from flowers of Calystegia hederacea in China. Int. J. Syst. Bacteriol. Garnier, M., M. Clerc and J.M. Bové. 1984. Growth and division of 40: 421–425. Spiroplasma citri: elongation of elementary helices. J. Bacteriol. 158: Hackett, K.J. and D.E. Lynn. 1985. Cell-assisted growth of a fastidious 23–28. spiroplasma. Science 230: 825–827. Garnier, M., X. Foissac, P. Gaurivaud, F. Laigret, J. Renaudin, C. Saillard Hackett, K.J., D.E. Lynn, D.L. Williamson, A.S. Ginsberg and R.F. Whit- and J.M. Bové. 2001. Mycoplasmas, plants, insect vectors: a matrimo- comb. 1986. Cultivation of the Drosophila sex-ratio Spiroplasma. Sci- nial triangle. C. R. Acad. Sci. III 324: 923–928. ence 232: 1253–1255. Genus I. Spiroplasma 681

Hackett, K.J. and T.B. Clark. 1989. Ecology of Spiroplasmas. In The Hurst, G.D.D., J.H.G. von der Schulenburg, T.M.O. Majerus, D. Bertrand, Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic I.A. Zakharov, J. Baungaard, W. Volkl, R. Stouthamer and M.E.N. Press, New York, pp. 113–200. Majerus. 1999. Invasion of one insect species, Adalia bipunctata, by two Hackett, K.J., R.F. Whitcomb, R.B. Henegar, A.G. Wagner, E.A. Clark, different male-killing bacteria. Insect Mol. Biol. 8: 133–139. J.G. Tully, F. Green, W.H. McKay, P. Santini, D.L. Rose, J.J. Ander- Hurst, G.D.D. and F.M. Jiggins. 2000. Male-killing bacteria in insects: mech- son and D.E. Lynn. 1990. Mollicute diversity in arthropod hosts. anisms, incidence, and implications. Emerg. Infect. Dis. 6: 329–336. Zentralbl. Bakteriol. Suppl. 20: 441–454. International Committee on Systematics of Bacteria. 1984. Minutes of Hackett, K.J., R.F. Whitcomb, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, the interim meeting. 30 August and 6 September 1982, Tokyo, Japan R.B. Henegar, T.B. Clark, E.A. Clark, M. Konai, J.R. Adams and D.L. Int. J. Syst. Bacteriol. 34: 361–365. Williamson. 1993. Spiroplasma insolitum sp. nov., a new species of International Committee on Systematics of Bacteria Subcommittee on group-I spiroplasma with an unusual DNA base composition. Int. J. the Taxonomy of Mollicutes. 1995. Revised minimum standards for Syst. Bacteriol. 43: 272–277. description of new species of the class Mollicutes (division Tenericutes). Hackett, K.J., R.H. Hackett, E.A. Clark, G.E. Gasparich, J.D. Pollack and Int J. Syst. Bacteriol 45: 605–612. R.F. Whitcomb. 1994. Development of the first completely defined Jacob, C., F. Nouzieres, S. Duret, J.M. Bové and J. Renaudin. 1997. Isola- medium for a spiroplasma, Spiroplasma clarkii strain CN-5. IOM Lett. tion, characterization, and complementation of a motility mutant of 3: 446–447. Spiroplasma citri. J. Bacteriol. 179: 4802–4810. Hackett, K.J. and R.F. Whitcomb. 1995. Cultivation of spiroplasmas in Jaenike, J., M. Polak, A. Fiskin, M. Helou and M. Minhas. 2007. Inter- undefined and defined media. In Molecular and Diagnostic Proce- specific transmission of endosymbiotic Spiroplasma by mites. Biol. dures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Aca- Lett. 3: 23–25. demic Press, San Diego, pp. 41–53. Jagoueix-Eveillard, S., F. Tarendeau, K. Guolter, J.L. Danet, J.M. Bové and Hackett, K.J., E.A. Clark, R.F. Whitcomb, M. Camp and J.G. Tully. 1996a. M. Garnier. 2001. Catharanthus roseus genes regulated differentially by Amended data on arginine utilization by Spiroplasma species. Int. J. mollicute infections. Mol. Plant Microbe Interact. 14: 225–233. Syst. Bacteriol. 46: 912–915. Jandhyam, H., C. R. Bates, T. E. Young, L. Beatti, G. E. Gasparich, F. E. Hackett, K.J., R.F. Whitcomb, T.B. Clark, R.B. Henegar, D.E. Lynn, A.G. French and L. B. Regassa. 2008. Global spiroplasma biodiversity in a Wagner, J.G. Tully, G.E. Gasparich, D.L. Rose, P. Carle, J.M. Bové, single host. Presented at the 17th Congress of International Organi- M. Konai, E.A. Clark, J.R. Adams and D.L. Williamson. 1996b. Spiro- zation for Mycoplasmology, Beijing, China. Abstract no. 206, p. 130. plasma leptinotarsae sp. nov., a mollicute uniquely adapted to its host, Jiggins, F.M., G.D. Hurst, C.D. Jiggins, J.H. von der Schulenburg and the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: M.E. Majerus. 2000. The butterfly Danaus chrysippus is infected by a Chrysomelidae). Int. J. Syst. Bacteriol. 46: 906–911. male-killing Spiroplasma bacterium. Parasitology 120: 439–446. Hackett, K.J., R.F. Whitcomb, F.E. French, J.G. Tully, G.E. Gasparich, Johansson, K.-E., M.U.K. Heldtander and B. Pettersson. 1998. Charac- D.L. Rose, P. Carle, J.M. Bové, R.B. Henegar, T.B. Clark, M. Konai, terization of mycoplasmas by PCR and sequence analysis with univer- E.A. Clark and D.L. Williamson. 1996c. Spiroplasma corruscae sp. nov., sal 16S rDNA primers. In Methods in Molecular Biology: Mycoplasma from a firefly beetle (Coleoptera: Lampyridae) and tabanid flies protocols, vol. 104 (edited by Miles and Nicholas). Humana Press, (Diptera: Tabanidae). Int. J. Syst. Bacteriol. 46: 947–950. Totawa, NJ, pp. 145–165. Hackett, K.J., J.J. Lipa, G.E. Gasparich, D.E. Lynn, M. Konai, M. Camp Johansson, K.-E. and B. Pettersson. 2002. Taxonomy of Mollicutes. In and R.F. Whitcomb. 1997. The spiroplasma motility inhibition test, a Molecular Biology and Pathogenicity of Mycoplasmas (edited by new method for determining intraspecific variation among Colorado Razin and Herrmann). Kluwer Academic/Plenum Publishers, potato beetle spiroplasmas. Int. J. Syst. Bacteriol. 47: 33–37. ­London, pp. 1–31. Haselkorn, T.S., T.A. Markow and N.A. Moran. 2009. Multiple introduc- Johnson, J.L. 1994. Similarity analysis of DNAs. In Methods for General tions of the Spiroplasma bacterial endosymbiont into Drosophila. Mol and Molecular Bacteriology (edited by Gerhardt, Murray, Wood and Ecol 18: 1294–1305. Krieg). ASM Press, Washington, D.C., pp. 656–682. Hélias, C., M. Vazeille-Falcoz, F. Le Goff, M.L. Abalain-Colloc, F. Rod- Jones, L.J., R. Carballido-Lopez and J. Errington. 2001. Control of cell hain, P. Carle, R.F. Whitcomb, D.L. Williamson, J.G. Tully, J.M. Bové shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell and C. Chastel. 1998. Spiroplasma turonicum sp. nov. from Haematopota 104: 913–922. horse flies (Diptera: Tabanidae) in France. Int. J. Syst. Bacteriol. 48: Joshi, B.D., M. Berg, J. Rogers, J. Fletcher and U. Melcher. 2005. 457–461. Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and Henning, K., S. Greiner-Fischer, H. Hotzel, M. Ebsen and D. Theegar- pSKU146 of S. kunkelii: implications for plasmid evolution. BMC ten. 2006. Isolation of Spiroplasma sp. from an Ixodes tick. Int. J. Med. Genomics 6: 175. Microbiol. 296 Suppl 40: 157–161. Junca, P., C. Saillard, J. Tully, O. Garcia-Jurado, J.R. Degorce-Dumas, C. Herren, J.K., I. Gordon, P. W. H. Holland and D. Smith. 2007. The but- Mouches, J.C. Vignault, R. Vogel, R. McCoy, R. Whitcomb, D. Wil- terfly Danaus chrysippus (Lepidoptera: Nymphalidae) in Kenya is vari- liamson, J. Latrille and J.M. Bové. 1980. Characterization of spiro- ably infected with respect to genotype and body size by a maternally plasmas isolated from insects and flowers in continental France, transmitted male-killing endosymbiont (Spiroplasma). Int. J. Trop. Corsica and Morocco. Proposals for a taxonomical classification of Ins. Sc.: 62–69. spiroplasmas. [transl. from. Fr.] C. R. Hebd. Des Seances Acad. Sci. Humphery-Smith, I., O. Grulet and C. Chastel. 1991a. Pathogenicity of Ser. D Sci. Nat. 290: 1209–1211. Spiroplasma taiwanense for larval Aedes aegypti mosquitoes. Med. Vet. Kageyama, D., H. Anbutsu, M. Watada, T. Hosokawa, M. Shimada and T. Entomol. 5: 229–232. Fukatsu. 2006. Prevalence of a non-male-killing spiroplasma in natural Humphery-Smith, I., O. Grulet, F. Le Goff and C. Chastel. 1991b. Spiro- populations of Drosophila hydei. Appl Environ Microbiol 72: 6667–6673. plasma (Mollicutes: Spiroplasmataceae) pathogenic for Aedes aegypti and Kersting, U. and C. Sengonca. 1992. Detection of insect vectors of the Anopheles stephensi (Diptera: Culicidae). J. Med. Entomol. 28: 219–222. citrus stubborn disease pathogen, Spiroplasma citri Saglio et al., in the Hung, S.H.Y., T.A. Chen, R.F. Whitcomb, J.G. Tully and Y.X. Chen. 1987. citrus growing area of south Turkey. J. Appl. Entomol. 113: 356–364. Spiroplasma culicicola sp. nov. from the salt-marsh mosquito Aedes sol- Killiny, N., M. Castroviejo and C. Saillard. 2005. Spiroplasma citri spiralin licitans. Int. J. Syst. Bacteriol. 37: 365–370. acts in vitro as a lectin binding to glycoproteins from its insect vector Hurst, G.D.D., H. Anbutsu, M. Kutsukake and T. Fukatsu. 2003. Hidden Circulifer haematoceps. Phytopathology 95: 541–548. from the host: Spiroplasma bacteria infecting Drosophila do not cause Killiny, N., B. Batailler, X. Foissac and C. Saillard. 2006. Identification of an immune response, but are suppressed by ectopic immune activa- a Spiroplasma citri hydrophilic protein associated with insect transmis- tion. Insect Mol. Biol. 12: 93–97. sibility. Microbiology 152: 1221–1230. 682 Family II. Spiroplasmataceae

Koerber, R.T., G.E. Gasparich, M.F. Frana and W.L. Grogan, Jr. 2005. lato and A. funestus mosquitoes reveals new species related to known Spiroplasma atrichopogonis sp. nov., from a ceratopogonid biting insect symbionts. Appl. Environ. Microbiol. 71: 7217–7223. midge. Int. J. Syst. Evol. Microbiol. 55: 289–292. Liss, A. and R.M. Cole. 1981. Spiroplasmavirus group I: isolation, Konai, M., R.F. Whitcomb, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, R.B. growth, and properties. Curr. Microbiol. 5: 357–362. Henegar, K.J. Hackett, T.B. Clark and D.L. Williamson. 1995. Spiro- Lundgren, J.G., R. M. Lehman and J. Chee-Sanford. 2007. Bacterial plasma velocicrescens sp. nov., from the vespid wasp Monobia quadridens. communities within digestive tracts of ground beetles (Coleoptera: Int. J. Syst. Bacteriol. 45: 203–206. Carabidae). Ann. Entomol. Soc. Am. 100: 275–282. Konai, M., E.A. Clark, M. Camp, A.L. Koeh and R.F. Whitcomb. 1996a. Maccheroni, W., J. L. Danet, S. Duret-Nurbel, J. M. Bové, M. Garnier Temperature ranges, growth optima, and growth rates of Spiroplasma and J. Renaudin. 2002. Cell shape determination in Spiroplasma citri: (Spiroplasmataceae, class Mollicutes) species. Curr. Microbiol. 32: 314– organization of mreB genes and effect of mreB1 disruption on insect 319. transmission and pathogenicity. Proceedings of the 15th Conference Konai, M., K.J. Hackett, D.L. Williamson, J.J. Lipa, J.D. Pollack, G.E. Gas- of the International Organization of Citrus Virologists (edited by parich, E.A. Clark, D.C. Vacek and R.F. Whitcomb. 1996b. Improved Duran-Vila, Milne and da Graça), Riverside, California, p. 443. cultivation systems for isolation of the Colorado potato beetle spiro- Madden, L.V. and L.R. Nault. 1983. Differential pathogenicity of corn plasma. Appl. Environ. Microbiol. 62: 3453–3458. stunting mollicutes to leafhopper vectors in Dalbulus and Baldulus Konai, M., R.F. Whitcomb, F.E. French, J.G. Tully, D.L. Rose, P. Carle, species. Phytopathology 73: 1608–1614. J.M. Bové, K.J. Hackett, R.B. Henegar, T.B. Clark and D.L. William- Majerus, T.M., J.H. Graf von der Schulenburg, M.E. Majerus and G.D. son. 1997. Spiroplasma litorale sp. nov., from tabanid flies (Tabanidae: Hurst. 1999. Molecular identification of a male-killing agent in the Diptera) in the southeastern United States. Int. J. Syst. Bacteriol. 47: ladybird Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). 359–362. Insect. Mol. Biol. 8: 551–555. Kotani, H., G.H. Butler and G.J. McGarrity. 1990. Malignant transforma- Maniloff, J. 1992. Phylogeny of mycoplasmas. In Mycoplasmas: Molecu- tion by Spiroplasma mirum. Zentralbl. Bakteriol. Suppl. 20: 145–152. lar Biology and Pathogenesis (edited by Maniloff, McElhaney, Finch Kürner, J., A.S. Frangakis and W. Baumeister. 2005. Cyro-electron and Baseman). American Society for Microbiology, Washington, tomography reveals the cytoskeletal structure of Spiroplasma mellif- D.C., pp. 549–559. erum. Science 307: 436–438. Marais, A., J.M. Bové, S.F. Dallo, J.B. Baseman and J. Renaudin. 1993. Kuroda, Y., Y. Shimada, B. Sakaguchi and K. Oishi. 1992. Effects of sex- Expression in Spiroplasma citri of an epitope carried on the G frag- ratio (SR)-spiroplasma infection on Drosophila primary embryonic ment of the cytadhesin P1 gene from Mycoplasma pneumoniae. J. Bac- cultured cells and on embryogenesis. Zool. Sci. 9: 283–291. teriol. 175: 2783–2787. Labarère, J. and G. Barroso. 1989. Lethal and mutation frequency Marais, A., J.M. Bové and J. Renaudin. 1996. Spiroplasma citri virus responses of Spiroplasma citri cells to UV irradiation. Mutat. Res. 210: SpV1-derived cloning vector: deletion formation by illegitimate and 135–141. homologous recombination in a spiroplasmal host strain which prob- Laigret, F., P. Gaurivaud and J.M. Bové. 1996. The unique organization ably lacks a functional recA gene. J. Bacteriol. 178: 862–870. of the rpoB region of Spiroplasma citri: a restriction and modification Markham, P.G., R. Townsend, M. Bar Joseph, M.J. Daniels, A. Plaskitt system gene is adjacent to rpoB. Gene 171: 95–98. and B.M. Meddins. 1974. Spiroplasmas are causal agents of citrus Lartigue, C., S. Duret, M. Garnier and J. Renaudin. 2002. New plas- little-leaf disease. Ann. Appl. Biol. 78: 49–57. mid vectors for specific gene targeting in Spiroplasma citri. Plasmid Markham, P.G., T.B. Clark and R.F. Whitcomb. 1983. Culture techniques 48: 149–159. for spiroplasmas from arthropods. In Methods in Mycoplasmology, vol. Le Dantec, L., M. Castroviejo, J.M. Bové and C. Saillard. 1998. Purifica- 2 (edited by Tully and Razin). Academic Press, New York, pp. 217–223. tion, cloning, and preliminary characterization of a Spiroplasma citri Mateos, M., S.J. Castrezana, B.J. Nankivell, A.M. Estes, T.A. Markow and ribosomal protein with DNA binding capacity. J. Biol. Chem. 273: N.A. Moran. 2006. Heritable endosymbionts of Drosophila. Genetics 24379–24386. 174: 363–376. Le Goff, F., M. Marjolet, J. Guilloteau, I. Humphery-Smith and C. Chas- Matsuo, K., J. Silke, K. Gramatikoff and W. Schaffner. 1994. The CpG- tel. 1990. Characterization and ecology of mosquito spiroplasmas specific methylase SssI has topoisomerase activity in the presence of from Atlantic biotopes in France. Ann. Parasitol. Hum. Comp. 65: Mg2+. Nucleic Acids Res. 22: 5354–5359. 107–110. McCoy, R.E., D.S. Williams and D.L. Thomas. 1979. Isolation of myco- Le Goff, F., I. Humphery-Smith, M. Leclercq and C. Chastel. 1991. Spiro- plasmas from flowers. Proceedings of the Republic of China-United plasmas from European Tabanidae. Med. Vet. Entomol. 5: 143–144. States Cooperative Science Seminar, Symposium series 1, National Le Goff, F., M. Marjolet, I. Humphery-Smith, M. Leclercq, C. Hélias, Science Council, Taipei, Taiwan, pp. 75–81. F. Suplisson and C. Chastel. 1993. Tabanid spiroplasmas from France: McElwain, M.C., D.K.F. Chandler, M.F. Barile, T.F. Young, V.V. Tryon, characterization, ecology and experimental study. Ann. Parasitol. J.W. Davis, J.P. Petzel, C.J. Chang, M.V. Williams and J.D. Pollack. Hum. Comp. 68: 150–153. 1988. Purine and pyrimidine metabolism in Mollicutes species. Int. J. Lee, I.M. and R.E. Davis. 1980. DNA homology among diverse spiro- Syst. Bacteriol. 38: 417–423. plasma strains representing several serological groups. Can. J. Micro- McIntosh, M.A., G. Deng, J. Zheng and R.V. Ferrell. 1992. Repetitive biol. 26: 1356–1363. DNA sequences. In Mycoplasmas: Molecular Biology and Pathogen- Liao, C.H. and T.A. Chen. 1977. Culture of corn stunt spiroplasma in a esis (edited by Maniloff, McElhaney, Finch and Baseman). American simple medium. Phytopathology 67: 802–807. Society for Microbiology, Washington, D.C., pp. 363–376. Liao, C.H., C.J. Chang and T.A. Chen. 1979. Spiroplasmastatic action of Melcher, U. and J. Fletcher. 1999. Genetic variation in Spiroplasma citri. plant tissue extracts. Proceedings of the R. O. C. U. S. Coop. Science Eur. J. Plant Pathol. 105: 519–533. Seminar Mycoplasma Diseases of Plants, Taipei, pp. 99–103. Miles, R.J. 1992. Catabolism in Mollicutes. J. Gen. Microbiol. 138: 1773– Liao, C.H. and T.A. Chen. 1981a. Deoxyribonucleic acid hybridiza- 1783. tion between Spiroplasma citri and the corn stunt spiroplasma. Curr. Montenegro, H., V.N. Solferini, L.B. Klaczko and G.D. Hurst. 2005. Microbiol. 5: 83–86. Male-killing Spiroplasma naturally infecting Drosophila melanogaster. Liao, C.H. and T.A. Chen. 1981b. In vitro susceptibility and resistance of Insect. Mol. Biol. 14: 281–287. two spiroplasmas to antibiotics. Phytopathology 71: 442–445. Montenegro, H., L. M. Hatadani, H. F. Medeiros and L.B. Klaczko. 2006. Lindh, J.M., O. Terenius and I. Faye. 2005. 16S rRNA gene-based identi- Male killing in three species of the tripunctata radiation of Drosophila fication of midgut bacteria from field-caught Anopheles gambiae sensu (Diptera: Drosophilidae). J. Zoo. Syst. Evol. Res. 44: 130–135. Genus I. Spiroplasma 683

Mouches, C., J.M. Bové, J. Albisetti, T.B. Clark and J.G. Tully. 1982a. Oishi, K., D.F. Poulson and D.L. Williamson. 1984. Virus-mediated A spiroplasma of serogroup IV causes a May-disease-like disorder of change in clumping properties of Drosophila SR spiroplasmas. Curr. honeybees in southwestern France. Microb. Ecol. 8: 387–399. Microbiol. 10: 153–158. Mouches, C., A. Menara, B. Geny, D. Charlemagne and J.M. Bové. Özbek, E., S.A. Miller, T. Meulia and S.A. Hogenhout. 2003. Infection 1982b. Synthesis of Spiroplasma citri protein specifically recognized by and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in rabbit immunoglobulin to rabbit actin. Rev. Infect. Dis. 4: S277. midgut and Malpighian tubules of the leafhopper Dalbulus maidis. J. Mouches, C., A. Menara, J.G. Tully and J.M. Bové. 1982c. Polyacrylam- Invertebr. Pathol. 82: 167–175. ide gel analysis of spiroplasmas proteins and its contribution to the Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2000. Updated taxonomy of spiroplasmas. Rev. Infect. Dis. 4 Suppl: S141–147. phylogenetic description of the Mycoplasma hominis cluster (Weisburg Mouches, C., J.M. Bové, J.G. Tully, D.L. Rose, R.E. McCoy, P. Carle- et al. 1989) based on 16S rDNA sequences. Int. J. Syst. Evol. Micro- Junca, M. Garnier and C. Saillard. 1983a. Spiroplasma apis, a new spe- biol. 50: 291–301. cies from the honey bee Apis mellifera. Ann. Microbiol. (Paris) 134A: Pickens, E.G., R.K. Gerloff and W. Burgdorfer. 1968. Spirochete from 383–397. the rabbit tick, Haemaphysalis leporispalustris (Packard). I. Isolation Mouches, C., T. Candresse, G.J. McGarrity and J.M. Bové. 1983b. Analy- and preliminary characterization. J. Bacteriol. 95: 291–299. sis of spiroplasma proteins: contribution to the taxonomy of group Pollack, J.D., M.C. McElwain, D. Desantis, J.T. Manolukas, J.G. Tully, IV spiroplasmas and the characterization of spiroplasma protein anti- C.J. Chang, R.F. Whitcomb, K.J. Hackett and M.V. Williams. 1989. gens. Yale J. Biol. Med. 56: 431–437. Metabolism of members of the Spiroplasmataceae. Int. J. Syst. Bacte- Mouches, C., G. Barroso, A. Gadeau and J.M. Bové. 1984a. Character- riol. 39: 406–412. ization of two cryptic plasmids from Spiroplasma citri and occurrence Pollack, J.D., M.V. Williams and R.N. McElhaney. 1997. The compara- of their DNA sequences among various spiroplasmas. Ann. Micro- tive metabolism of the mollicutes (mycoplasmas): the utility for taxo- biol. (Paris) 135A: 17–24. nomic classification and the relationship of putative gene annotation Mouches, C., J. M. Bové, J. G. Tully, D. L. Rose, R. E. McCoy, P. Carle- and phylogeny to enzymatic function in the smallest free-living cells. Junca, M. Garnier and C. Saillard. 1984b. In Validation of the pub- Crit. Rev. Microbiol. 23: 269–354. lication of new names and new combinations previously effectively Pollack, J.D. 2002a. The necessity of combining genomic and enzymatic published outside the IJSB. List no. 13. Int. J. Syst. Bacteriol. 34: data to infer metabolic function and pathways in the smallest bacte- 91–92. ria: amino acid, purine and pyrimidine metabolism in mollicutes. Moya-Raygoza, G., S.A. Hogenhout and L.R. Nault. 2007a. Habitat of Front. Biosci. 7: d1762–1781. the corn leafhopper (Hemiptera: Cicadellidae) during the dry (win- Pollack, J.D. 2002b. Central carbohydrate pathways: Metabolic flexibil- ter) season in Mexico. Environ Entomol 36: 1066–1072. ity and the extra role of some “housekeeping enzymes”. In Molecu- Moya-Raygoza, G., V. Palomera-Avalos and C. Galaviz-Mejia. 2007b. lar Biology and Pathogenicity of Mycoplasmas (edited by Razin and Field overwintering biology of Spiroplasma kunkelii (Mycoplasmatales: Herrmann). Kluwer Academic/Plenum Publishers, New York, pp. Spiroplasmataceae) and its vector Dalbulus maidis (Hemiptera: Cicadel- 163–199. lidae). Ann. Appl. Biol. 151: 373–379. Pool, J.E., A. Wong and C.F. Aquadro. 2006. Finding of male-killing Nakamura, K., H. Ueno and K. Miura. 2005. Prevalence of inherited Spiroplasma infecting Drosophila melanogaster in Africa implies transat- male-killing microorganisms in Japanese populations of ladybird lantic migration of this endosymbiont. Heredity 97: 27–32. beetle Harmonia axyridis (Coleoptera: Coccinellidae). Ann. Ent. Soc. Poulson, D.F. and B. Sakaguchi. 1961. Nature of “sex-ratio” agent in Am. 98: 96–99. Drosophila. Science 133: 1489–1490. Nault, L.R. and O.E. Bradfute. 1979. Corn stunt: involvement of a Pyle, L.E. and L.R. Finch. 1988. A physical map of the genome of complex of leafhopper-borne pathogens. In Leafhopper Vectors Mycoplasma mycoides subspecies mycoides Y with some functional loci. and Plant Disease Agents (edited by Maramorosch and Harris). Aca- Nucleic Acids Res. 16: 6027–6039. demic Press, New York, pp. 561–586. Rahimian, H. and D.J. Gumpf. 1980. Deoxyribonucleic acid relation- Nault, L.R., L.V. Madden, W.E. Styer, B.W. Triplehorn, G.F. Shambaugh ship between Spiroplasma citri and the corn stunt spiroplasma. Int. J. and S.E. Heady. 1984. Pathogenicity of corn stunt spiroplasma and Syst. Bacteriol. 30: 605–608. maize bushy stunt mycoplasma to their vector, Dalbulus longulus. Phy- Ranhand, J.M., W.O. Mitchell, T.J. Popkin and R.M. Cole. 1980. Cova- topathology 74: 977–979. lently closed circular deoxyribonucleic acids in spiroplasmas. J. Bac- Navas-Castillo, J., F. Laigret, J.G. Tully and J.M. Bové. 1992. The mol- teriol. 143: 1194–1199. licute Acholeplasma florum possesses a gene of phosphoenolpyruvate- Razin, S. 1985. Molecular biology and genetics of mycoplasmas (Mol- sugar phosphotransferase system and it uses UGA as tryptophan licutes). Microbiol. Rev. 49: 419–455. codon. C. R. Acad. Sci. Ser. III Life Sci. 315: 43–48. Regassa, L.B., K.M. Stewart, A.C. Murphy, F.E. French, T. Lin and R.F. Nunan, L.M., C.R. Pantoja, M. Salazar, F. Aranguren and D.V. Light- Whitcomb. 2004. Differentiation of group VIII Spiroplasma strains ner. 2004. Characterization and molecular methods for detection with sequences of the 16S–23S rDNA intergenic spacer region. Can. of a novel spiroplasma pathogenic to Penaeus vannamei. Dis. Aquat. J. Microbiol. 50: 1061–1067. Organ. 62: 255–264. Regassa, L.B. and G.E. Gasparich. 2006. Spiroplasmas: evolutionary Nunan, L.M., D.V. Lightner, M.A. Oduori and G.E. Gasparich. 2005. Spiro- relationships and biodiversity. Front. Biosci. 11: 2983–3002. plasma penaei sp. nov., associated with mortalities in Penaeus vannamei, Renaudin, J., M.C. Pascarel, M. Garnier, P. Carle-Junca and J.M. Bové. Pacific white shrimp. Int. J. Syst. Evol. Microbiol. 55: 2317–2322. 1984a. SpV4, a new Spiroplasma virus with circular, single-stranded Nur, I., M. Szyf, A. Razin, G. Glaser, S. Rottem and S. Razin. 1985. Pro- DNA. Ann. Virol. 135E: 163–168. caryotic and eucaryotic traits of DNA methylation in spiroplasmas Renaudin, J., M.C. Pascarel, M. Garnier, P. Carle and J.M. Bové. 1984b. (mycoplasmas). J. Bacteriol. 164: 19–24. Characterization of spiroplasma virus group 4 (SV4). Isr. J. Med. Sci. Nur, I., G. Glaser and S. Razin. 1986. Free and integrated plasmid DNA 20: 797–799. in spiroplasmas. Curr. Microbiol. 14: 169–176. Renaudin, J., M.C. Pascarel, C. Saillard, C. Chevalier and J.M. Bové. Nur, I., D.J. LeBlanc and J.G. Tully. 1987. Short, interspersed, and repet- 1986. Chez les spiroplasmes le codon UGA n’est pas non-sens et sem- itive DNA sequences in Spiroplasma species. Plasmid 17: 110–116. ble coder pour le tryptophane. C. R. Acad. Sci. Ser. III 303: 539–540. Oduori, M.A., J.J. Lipa and G.E. Gasparich. 2005. Spiroplasma leucomae Renaudin, J. and J.M. Bové. 1994. SpV1 and SpV4, spiroplasma viruses sp. nov., isolated in Poland from white satin moth (Leucoma salicis L.) with circular, single-stranded DNA genomes, and their contribution to larvae. Int. J. Syst. Evol. Microbiol. 55: 2447–2450. the molecular biology of spiroplasmas. Adv. Virus Res. 44: 429–463. 684 Family II. Spiroplasmataceae

Renaudin, J., A. Marais, E. Verdin, S. Duret, X. Foissac, F. Laigret and viral DNA sequences into host chromosomal and extrachromosomal J.M. Bové. 1995. Integrative and free Spiroplasma citri oriC plasmids: DNA. Appl. Environ. Microbiol. 61: 3950–3959. Expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri. Shaevitz, J.W., J.Y. Lee and D.A. Fletcher. 2005. Spiroplasma swim by a J. Bacteriol. 177: 2870–2877. processive change in body helicity. Cell 122: 941–945. Renaudin, J. 2002. Extrachromosomal elements and gene transfer. Sher, T., A. Yamin, M. Matzliach, S. Rottem and R. Gallily. 1990a. Partial In Molecular Biology and Pathogenicity of Mycoplasmas (edited biochemical characterization of spiroplasma membrane component by Razin and Herrmann). Academic/Plenum Press, New York, pp. inducing tumor necrosis factor alpha. Anticancer Drugs 1: 83–87. 347–370. Sher, T., A. Yamin, S. Rottem and R. Gallily. 1990b. In vitro induction Renbaum, P., D. Abrahamove, A. Fainsod, G.G. Wilson, S. Rottem and of tumor necrosis factor alpha, tumor cytolysis, and blast transfor- A. Razin. 1990. Cloning, characterization, and expression in Escheri- mation by Spiroplasma membranes. J. Natl. Cancer Inst. 82: 1142– chia coli of the gene coding for the CpG DNA methylase from Spiro- 1145. plasma sp. strain MQ1(M-SssI). Nucleic Acids Res. 18: 1145–1152. Simoneau, P. and J. Labarère. 1991. Evidence for the presence of two Renbaum, P. and A. Razin. 1992. Mode of action of the Spiroplasma CpG distinct membrane ATPases in Spiroplasma citri. J. Gen. Microbiol. methylase M-SssI. FEBS Lett. 313: 243–247. 137: 179–185. Ricard, B., M. Garnier and J.M. Bové. 1982. Characterization of spiro- Skripal, I.G. 1974. On improvement of taxonomy of the class Mollicutes plasmal virus 3 from spiroplasmas and discovery of a new spiroplas- and establishment in the order Mycoplasmatales of the new family mal virus (SpV4). Rev. Infect. Dis. 4: S275. Spiroplasmataceae fam. nov. Mikrobiol. Zh. (Kiev). 36: 462–467. Rodwell, A.W. and R.F. Whitcomb. 1983. Methods of direct and indirect Skripal, I.G. 1983. Revival of the name Spiroplasmataceae fam. nov., nom. measurement of mycoplasma growth. In Methods in Mycoplasmol- rev., omitted from the 1980 Approved Lists of Bacterial Names. Int. ogy, vol. 1 (edited by Razin and Tully). Academic Press, New York, J. Syst. Bacteriol. 33: 408. pp. 185–196. Sokolova, M.I., N.S. Zinkevich and I.A. Zakharov. 2002. Bacteria in ova- Rogers, M.J., J. Simmons, R.T. Walker, W.G. Weisburg, C.R. Woese, R.S. rioles of females from maleless families of ladybird beetles Adalia Tanner, I.M. Robinson, D.A. Stahl, G. Olsen, R.H. Leach and J. Maniloff. bipunctata L. (Coleoptera: Coccinellidae) naturally infected with 1985. Construction of the mycoplasma evolutionary tree from 5S rRNA Rickettsia, Wolbachia, and Spiroplasma. J. Invertebr. Pathol. 79: 72–79. sequence data. Proc. Natl. Acad. Sci. U. S. A. 82: 1160–1164. Stackebrandt, E., W. Frederiksen, G.M. Garrity, P.A. Grimont, P. Kämp- Rogers, M.J., A.A. Steinmetz and R.T. Walker. 1987. Organization and fer, M.C. Maiden, X. Nesme, R. Rosselló-Mora, J. Swings, H.G. Trüper, structure of tRNA genes in Spiroplasma melliferum. Isr. J. Med. Sci. 23: L. Vauterin, A.C. Ward and W.B. Whitman. 2002. Report of the ad 357–360. hoc committee for the re-evaluation of the species definition in bac- Rose, D.L., J.G. Tully, J.M. Bové and R.F. Whitcomb. 1993. A test for teriology. Int. J. Syst. Evol. Microbiol. 52: 1043–1047. measuring growth responses of Mollicutes to serum and polyoxyethyl- Stamburski, C., J. Renaudin and J.M. Bove. 1991. First step toward a ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. virus-derived vector for gene cloning and expression in spiroplas- Rosengarten, R. and K.S. Wise. 1990. Phenotypic switching in myco- mas, organisms which read UGA as a tryptophan codon: synthesis plasmas: phase variation of diverse surface lipoproteins. Science 247: of chloramphenicol acetyltransferase in Spiroplasma citri. J. Bacteriol. 315–318. 173: 2225–2230. Rosselló-Mora, R. and R. Amann. 2001. The species concept for prokary- Stephens, M.A. 1980. Studies on Spiroplasma viruses. PhD thesis, Univer- otes. FEMS Microbiol. Rev 25: 39–67. sity of East Anglia, Norwich, UK. Saglio, P., D. Laflèche, C. Bonissol and J.M. Bové. 1971. Isolation, culture Stevens, C., A.Y. Tang, E. Jenkins, R.L. Goins, J.G. Tully, D.L. Rose, M. and electronmicroscopy of mycoplasma-like structures associated Konai, D.L. Williamson, P. Carle, J. Bove, K.J. Hackett, F.E. French, with stubborn disease of citrus and their comparison with structures J. Wedincamp, R.B. Henegar and R.F. Whitcomb. 1997. Spiroplasma observed in citrus plants affected by greening disease. [transl. from lampyridicola sp. nov., from the firefly beetle Photuris pennsylvanicus. Fr.] Physiol. Vég. 9: 569–582. Int. J. Syst. Bacteriol. 47: 709–712. Saglio, P., M. L’Hospital, D. Laflèche, G. Dupont, J.M. Bové, J.G. Tully Summers, C.G., A. S. Newton and D.C. Opgenorth. 2004. Overwinter- and E.A. Freundt. 1973. Spiroplasma citri gen. and sp. nov.: a myco- ing of corn leafhopper, Dalbulus maidis (Homoptera: Cicadellidae), plasma-like organism associated with stubborn disease of citrus. Int. and Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae) in Cali- J. Syst. Bacteriol. 23: 191–204. fornia’s San Joaquin Valley. Environ. Entomol. 33: 1644–1651. Saglio, P.H.M. and R.F. Whitcomb. 1979. Diversity of wall-less prokary- Swofford, D.L. 1998. PAUP: Phylogenetic analysis using parsimony and otes in plant vascular tissue, fungi and invertebrate animals. In The other methods, 4 edn. Sinauer Associates, Sunderland, MA. Mycoplasmas, vol. 3 (edited by Whitcomb and Tully). Academic Taroura, S., Y. Shimada, Y. Sakata, T. Miyama, H. Hiraoka, M. Watanabe, Press, New York, pp. 1–36. K. Itamoto, M. Okuda and H. Inokuma. 2005. Detection of DNA Saillard, C. and J.M. Bové. 1983. Application of ELISA to spiroplasma of ‘Candidatus Mycoplasma haemominutum’ and Spiroplasma sp. in detection and classification. In Methods in Mycoplasmology, vol. 1 unfed ticks collected from vegetation in Japan. J. Vet. Med. Sci. 67: (edited by Razin and Tully). Academic Press, New York, pp. 471–476. 1277–1279. Saillard, C., J.C. Vignault, J.M. Bové, A. Raie, J.G. Tully, D.L. Williamson, Tinsley, M.C. and M.E. Majerus. 2006. A new male-killing parasitism: A. Fos, M. Garnier, A. Gadeau, P. Carle and R.F. Whitcomb. 1987. Spiroplasma bacteria infect the ladybird beetle Anisosticta novemdecim- Spiroplasma phoeniceum sp. nov., a new plant-pathogenic species from punctata (Coleoptera: Coccinellidae). Parasitology 132: 757–765. Syria. Int. J. Syst. Bacteriol. 37: 106–115. Tinsley, M.C. and M.E. Majerus. 2007. Small steps or giant leaps for Saillard, C., C. Chevalier and J.M. Bové. 1990. Structure and organiza- male-killers? Phylogenetic constraints to male-killer host shifts. BMC tion of the spiralin gene. Zentralbl. Bakteriol. Suppl. 20: 897–901. Evol. Biol. 7: 238. Saillard, C., P. Carle, S. Duret-Nurbel, R. Henri, N. Killiny, S. Carrere, Townsend, R., P.G. Markham, K.A. Plaskitt and M.J. Daniels. 1977. Isola- J. Gouzy, J.M. Bové, J. Renaudin and X. Foissac. 2008. The abundant tion and characterization of a nonhelical strain of Spiroplasma citri. J. extrachromosomal DNA content of the Spiroplasma citri GII3–3X Gen. Microbiol. 100: 15–21. genome. BMC Genomics 9: 195. Townsend, R., D.B. Archer and K.A. Plaskitt. 1980a. Purification and Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new preliminary characterization of Spiroplasma fibrils. J. Bacteriol. 142: method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 694–700. 406–425. Townsend, R., J. Burgess and K.A. Plaskitt. 1980b. Morphology and Sha, Y., U. Melcher, R.E. Davis and J. Fletcher. 1995. Resistance of Spiro- ultrastructure of helical an nonhelical strains of Spiroplasma citri. J. plasma citri lines to the virus SVTS2 is associated with integration of Bacteriol. 142: 973–981. Genus I. Spiroplasma 685

Trachtenberg, S. and R. Gilad. 2001. A bacterial linear motor: cellular and Wang, W., W. Gu, Z. Ding, Y. Ren, J. Chen and Y. Hou. 2005. A novel molecular organization of the contractile cytoskeleton of the helical Spiroplasma pathogen causing systemic infection in the crayfish Pro- bacterium Spiroplasma melliferum BC3. Mol. Microbiol. 41: 827–848. cambarus clarkii (Crustacea: Decapod), in China. FEMS Microbiol. Trachtenberg, S., S.B. Andrews and R.D. Leapman. 2003a. Mass distri- Lett. 249: 131–137. bution and spatial organization of the linear bacterial motor of Spiro- Wang, W., W. Gu, G.E. Gasparich, K. Bi, J. Ou, Q. Meng, T. Liang, plasma citri R8A2. J. Bacteriol. 185: 1987–1994. Q. Feng, J. Zhang and Y. Zhang. 2010. Spiroplasma eriocheiris sp. nov., Trachtenberg, S., R. Gilad and N. Geffen. 2003b. The bacterial linear a novel species associated with mortalities in Eriocheiris sinensis, motor of Spiroplasma melliferum BC3: from single molecules to swim- Chinese mitten crab. Int. J. Syst. Evol. Microbiol. ijs.0.020529-Ov1- ming cells. Mol. Microbiol. 47: 671–697. ijs.0.020529-0. Trachtenberg, S. 2004. Shaping and moving a Spiroplasma. J. Mol. Micro- Wayadande, A.C. and J. Fletcher. 1995. Transmission of Spiroplasma citri biol. Biotechnol. 7: 78–87. lines and their ability to cross gut and salivary gland barriers within Trachtenberg, S. 2006. The cytoskeleton of Spiroplasma: a complex lin- the leafhopper vector Circulifer tenellus. Phytopathology 85: 1256– ear motor. J. Mol. Microbiol. Biotechnol. 11: 265–283. 1259. Trachtenberg, S., L.M. Dorward, V.V. Speransky, H. Jaffe, S.B. Andrews Wayadande, A.C. and J. Fletcher. 1998. Development and use of an and R.D. Leapman. 2008. Structure of the cytoskeleton of Spiroplasma established cell line of the leafhopper Circulifer tenellus to characterize melliferum BC3 and its interactions with the cell membrane. J. Mol. Spiroplasma citri-vector interactions. J. Invertebr. Pathol. 72: 126–131. Biol. 378: 778–789. Wayne, L.G., D.J. Brenner, R.R. Colwell, P.A.D. Grimont, O. Kandler, Tully, J.G., R.F. Whitcomb, H.F. Clark and D.L. Williamson. 1977. Patho- M.I. Krichevsky, L.H. Moore, W.E.C. Moore, R.G.E. Murray, E. Stack- genic mycoplasmas: cultivation and vertebrate pathogenicity of a new ebrandt, M.P. Starr and H.G. Trüper. 1987. Report of the ad hoc com- Spiroplasma. Science 195: 892–894. mittee on the reconciliation of approaches to bacterial systematics. Tully, J.G., D.L. Rose, O. Garciajurado, J.C. Vignault, C. Saillard, J.M. Int. J. Syst. Bacteriol. 37: 463–464. Bové, R.E. McCoy and D.L. Williamson. 1980. Serological analysis of Weisburg, W.G., J.G. Tully, D.L. Rose, J.P. Petzel, H. Oyaizu, D. Yang, L. a new group of spiroplasmas. Curr. Microbiol. 3: 369–372. Mandelco, J. Sechrest, T.G. Lawrence, J. Van Etten, J. Maniloff and Tully, J.G., D.L. Rose, C.E. Yunker, J. Cory, R.F. Whitcomb and D.L. C.R. Woese. 1989. A phylogenetic analysis of the mycoplasmas: basis ­Williamson. 1981. Helical mycoplasmas (spiroplasmas) from Ixodes for their classification. J. Bacteriol. 171: 6455–6467. ticks. Science 212: 1043–1045. Whitcomb, R.F., J.G. Tully, J.M. Bové and P. Saglio. 1973. Spiroplasmas Tully, J.G., R.F. Whitcomb, D.L. Rose and J.M. Bové. 1982. Spiroplasma and acholeplasmas: multiplication in insects. Science 182: 1251– mirum, a new species from the rabbit tick (Haemaphysalis leporispalus- 1253. tris). Int. J. Syst. Bacteriol. 32: 92–100. Whitcomb, R.F. and D.L. Williamson. 1979. Pathogenicity of mycoplas- Tully, J.G., D.L. Rose, E. Clark, P. Carle, J.M. Bové, R.B. Henegar, R.F. mas for arthropods. Zentralbl. Bakteriol. Orig. A 245: 200–221. Whitcomb, D.E. Colflesh and D.L. Williamson. 1987. Revised group Whitcomb, R.F., J. G. Tully, P. McCawley and D.L. Rose. 1982. Applica- classification of the genus Spiroplasma (class Mollicutes), with pro- tion of the growth-inhibition test to Spiroplasma taxonomy. Int. J. Syst. posed new groups XII to XXIII. Int. J. Syst. Bacteriol. 37: 357–364. Bacteriol. 32: 387–394. Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised tax- Whitcomb, R.F. 1983. Culture media for spiroplasmas. In Methods in onomy of the class Mollicutes - proposed elevation of a monophyletic Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, cluster of arthropod-associated mollicutes to ordinal rank (Entomoplas- New York, pp. 147–158. matales ord. nov.), with provision for familial rank to separate species Whitcomb, R.F., T.A. Chen, D.L. Williamson, C. Liao, J.G. Tully, J.M. with nonhelical morphology (Entomoplasmataceae fam. nov.) from Bové, C. Mouches, D.L. Rose, M.E. Coan and T.B. Clark. 1986. Spiro- helical species (Spiroplasmataceae), and emended descriptions of the plasma kunkelii sp. nov.: characterization of the etiologic agent of order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. corn stunt disease. Int. J. Syst. Bacteriol. 36: 170–178. 43: 378–385. Whitcomb, R.F. and K.J. Hackett. 1987. Cloning by limiting dilution in Tully, J.G., D.L. Rose, C.E. Yunker, P. Carle, J.M. Bové, D.L. Williamson liquid media: an improved alternative for cloning mollicute species. and R.F. Whitcomb. 1995. Spiroplasma ixodetis sp. nov., a new species Isr. J. Med. Sci. 23: 517. from Ixodes pacificus ticks collected in Oregon. Int. J. Syst. Bacteriol. Whitcomb, R.F. 1989. The biology of Spiroplasma kunkelii. In The Myco- 45: 23–28. plasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, Van den Ent, F., L. A. Amos and J. Löwe. 2001. Prokaryotic origin of the New York, pp. 487–544. actin cytoskeleton. Nature: 39–44. Whitcomb, R.F., K. J. Hackett, J. G. Tully, E. A. Clark, F. E. French, R. B. Vaughn, E.E. and W.M. de Vos. 1995. Identification and characteriza- Henegar, D. L. Rose and A.G. Wagner. 1990. Tabanid spiroplasmas tion of the insertion element IS1070 from Leuconostoc lactis NZ6009. as a model for mollicute biogeography. Zentrabl. Bakteriol. Suppl.: Gene 155: 95–100. 931–934. Vazeille-Falcoz, M., C. Hélias, F. Le Goff, F. Rodhain and C. Chastel. Whitcomb, R.F., C. Chastel, M. Abalain-Colloc, C. Stevens, J.G. Tully, 1997. Three spiroplasmas isolated from Haematopota sp. (Diptera: D.L. Rose, P. Carle, J.M. Bové, R.B. Henegar, K.J. Hackett, T.B. Clark, Tabanidae) in France. J. Med. Entomol. 34: 238–241. M. Konai and D.L. Williamson. 1993a. Spiroplasma cantharicola sp. Veneti, Z., J.K. Bentley, T. Koana, H.R. Braig and G.D.D. Hurst. 2005. A nov., from cantharid beetles (Coleoptera: Cantharidae). Int. J. Syst. functional dosage compensation complex required for male killing Bacteriol. 43: 421–424. in Drosophila. Science 307: 1461–1463. Whitcomb, R.F., J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, R.B. Henegar, Wada, H. and R.R. Netz. 2007. Model for self-propulsive helical fila- K.J. Hackett, T.B. Clark, M. Konai, J. Adams and D.L. Williamson. ments: kink-pair propagation. Phys. Rev. Lett. 99: 108102. 1993b. Spiroplasma monobiae sp. nov. from the vespid wasp Monobia Wang, W., L. Rong, W. Gu, K. Du and J. Chen. 2003. Study on experi- quadridens (Hymenoptera, Vespidae). Int. J. Syst. Bacteriol. 43: 256– mental infections of Spiroplasma from the Chinese mitten crab in cray- 260. fish, mice and embryonated chickens. Res. Microbiol. 154: 677–680. Whitcomb, R.F., J.C. Vignault, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, Wang, W., J. Chen, K. Du and Z. Xu. 2004a. Morphology of spiroplasmas K.J. Hackett, R.B. Henegar, M. Konai and D.L. Williamson. 1993c. in the Chinese mitten crab Eriocheir sinensis associated with tremor Spiroplasma clarkii sp. nov. from the green June beetle (Coleoptera, disease. Res Microbiol 155: 630–635. Scarabaeidae). Int. J. Syst. Bacteriol. 43: 261–265. Wang, W., B. Wen, G.E. Gasparich, N. Zhu, L. Rong, J. Chen and Z. Xu. Whitcomb, R.F., G.E. Gasparich, F.E. French, J.G. Tully, D.L. Rose, P. 2004b. A Spiroplasma associated with tremor disease in the Chinese Carle, J.M. Bové, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams, mitten crab (Eriocheir sinensis). Microbiology 150: 3035–3040. T.B. Clark and D.L. Williamson. 1996. Spiroplasma syrphidicola sp. 686 Family II. Spiroplasmataceae

nov., from a syrphid fly (Diptera: Syrphidae). Int. J. Syst. Bacteriol. C. Chastel and F.E. French. 1998. Revised group classification of the 46: 797–801. genus Spiroplasma. Int. J. Syst. Bacteriol. 48: 1–12. Whitcomb, R.F., F. E. French, J. G. Tully, P. Carle, R. Henegar, K. J. Williamson, D.L., B. Sakaguchi, K.J. Hackett, R.F. Whitcomb, J.G. Tully, Hackett, G. E. Gasparich and D.L. Williamson. 1997a. Spiroplasma P. Carle, J.M. Bové, J.R. Adams, M. Konai and R.B. Henegar. 1999. species, groups, and subgroups from North American Tabanidae. Spiroplasma poulsonii sp. nov., a new species associated with male- Curr. Microbiol. 35: 287–293. lethality in Drosophila willistoni, a neotropical species of fruit fly. Int. J. Whitcomb, R.F., F.E. French, J.G. Tully, G.E. Gasparich, D.L. Rose, P. Syst. Bacteriol. 49: 611–618. Carle, J. Bové, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams, T.B. Woese, C.R., J. Maniloff and L.B. Zablen. 1980. Phylogenetic analysis of Clark and D.L. Williamson. 1997b. Spiroplasma chrysopicola sp. nov., the mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 77: 494–498. Spiroplasma gladiatoris sp. nov., Spiroplasma helicoides sp. nov., and Spiro- Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221–271. plasma tabanidicola sp. nov., from tabanid (Diptera: Tabanidae) flies. Wolgemuth, C.W. and N.W. Charon. 2005. The kinky propulsion of Int. J. Syst. Bacteriol. 47: 713–719. Spiroplasma. Cell 122: 827–828. Whitcomb, R.F., F.E. French, J.G. Tully, D.L. Rose, P.M. Carle, J.M. Bove, Wróblewski, H., K.E. Johansson and S. Hjérten. 1977. Purification and E.A. Clark, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams and characterization of spiralin, the main protein of the Spiroplasma citri D.L. Williamson. 1997c. Spiroplasma montanense sp. nov., from Hybomi- membrane. Biochim. Biophys. Acta 465: 275–289. tra horseflies at northern latitudes in north America. Int. J. Syst. Bac- Wróblewski, H., S. Nyström, A. Blanchard and A. Wieslander. 1989. teriol. 47: 720–723. Topology and acylation of spiralin. J. Bacteriol. 171: 5039–5047. Whitcomb, R.F., J. G. Tully, G. E. Gasparich, L. B. Regassa, D. L. Wil- Wróblewski, H., D. Robic, D. Thomas and A. Blanchard. 1984. Com- liamson and F.E. French. 2007. Spiroplasma species in the Costa Rican parison of the amino acid compositions and antigenic properties of highlands: implications for biogeography and biodiversity. Biodivers. spiralins purified from the plasma membranes of different spiroplas- Conserv. 16: 3877–3894. mas. Ann. Microbiol. (Paris) 135A: 73–82. Williamson, D.L. 1969. The sex ratio spirochete in Drosophila robusta. Ye, F., F. Laigret, J.C. Whitley, C. Citti, L.R. Finch, P. Carle, J. Renaudin Jpn. J. Genet. 44: 36–41. and J.M. Bové. 1992. A physical and genetic map of the Spiroplasma Williamson, D.L. 1974. Unusual fibrils from the spirochete-like sex ratio citri genome. Nucleic Acids Res. 20: 1559–1565. organism. J. Bacteriol. 117: 904–906. Ye, F., F. Laigret and J.M. Bové. 1994a. A physical and genomic map Williamson, D.L. and R.F. Whitcomb. 1974. Helical wall-free prokary- of the prokaryote Spiroplasma melliferum and its comparison with the otes in Drosophila, leafhoppers and plants. Colloq. Inst. Natl. Santé Spiroplasma citri map. C. R. Acad. Sci. Ser. III 317: 392–398. Rech. Med. 33: 283–290. Ye, F., J. Renaudin, J.M. Bové and F. Laigret. 1994b. Cloning and Williamson, D.L. and R.F. Whitcomb. 1975. Plant mycoplasmas: a culti- sequencing of the replication origin (oriC) of the Spiroplasma citri vable spiroplasma causes corn stunt disease. Science 188: 1018–1020. chromosome and construction of autonomously replicating artificial Williamson, D.L., R. F. Whitcomb and J.G. Tully. 1978. The Spiroplasma plasmids. Curr. Microbiol. 29: 23–29. deformation test, a new serological method. Curr. Microbiol. 1: 203– Ye, F., F. Laigret, P. Carle and J.M. Bové. 1995. Chromosomal heteroge- 207. neity among various strains of Spiroplasma citri. Int. J. Syst. Bacteriol. Williamson, D.L., D.I. Blaustein, R.J.C. Levine and M.J. Elfvin. 1979a. 45: 729–734. Anti-actin-peroxidase staining of the helical wall-free prokaryote Ye, F., U. Melcher, J.E. Rascoe and J. Fletcher. 1996. Extensive chromo- Spiroplasma citri. Curr. Microbiol. 2: 143–145. some aberrations in Spiroplasma citri strain BR3. Biochem. Genet. 34: Williamson, D.L., J. G. Tully and R.F. Whitcomb. 1979b. Serological 269–286. relationships of spiroplasmas as shown by combined deformation Yogev, D., R. Rosengarten, R. Watson-McKown and K.S. Wise. 1991. and metabolism inhibition tests. Int. J. Syst. Bacteriol. 29: 345–351. Molecular basis of Mycoplasma surface antigenic variation: a novel set Williamson, D.L. and D.F. Poulson. 1979. Sex ratio organisms (spiro- of divergent genes undergo spontaneous mutation of periodic cod- plasmas) of Drosophila. In The Mycoplasmas, vol. 3 (edited by Whit- ing regions and 5¢ regulatory sequences. EMBO J. 10: 4069–4079. comb and Tully). Academic Press, New York, pp. 175–208. Yu, J., A.C. Wayadande and J. Fletcher. 2000. Spiroplasma citri surface Williamson, D.L. and R.F. Whitcomb. 1983. Special serological tests for protein P89 implicated in adhesion to cells of the vector Circulifer spiroplasma identification. In Methods in Mycoplasmology, vol. 2 tenellus. Phytopathology 90: 716–722. (edited by Razin and Tully). Academic Press, New York, pp. 249–259. Zaaria, A., C. Fontenelle, M. Le Henaff and H. Wróblewski. 1990. Anti- Williamson, D.L., P.R. Brink and G.W. Zieve. 1984. Spiroplasma fibrils. Isr. genic relatedness between the spiralins of Spiroplasma citri and Spiro- J. Med. Sci. 20: 830–835. plasma melliferum. J. Bacteriol. 172: 5494–5496. Williamson, D.L., J. G. Tully and R.F. Whitcomb. 1989. The genus Spiro- Zbinden, M. and M.A. Cambon-Bonavita. 2003. Occurrence of Defer- plasma. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). ribacterales and Entomoplasmatales in the deep-sea Alvinocarid shrimp Academic Press, San Diego, pp. 71–111. Rimicaris exoculata gut. FEMS Microbiol. Ecol. 46: 23–30. Williamson, D.L., J. Renaudin and J.M. Bové. 1991. Nucleotide Zhao, Y., R.W. Hammond, R. Jomantiene, E.L. Dally, I.M. Lee, H. Jia, sequence of the Spiroplasma citri fibril protein gene. J. Bacteriol. 173: H. Wu, S. Lin, P. Zhang, S. Kenton, F.Z. Najar, A. Hua, B.A. Roe, 4353–4362. J. Fletcher and R.E. Davis. 2003. Gene content and organization Williamson, D.L., J.G. Tully, L. Rosen, D.L. Rose, R.F. Whitcomb, M.L. of an 85-kb DNA segment from the genome of the phytopatho- Abalain-Colloc, P. Carle, J.M. Bové and J. Smyth. 1996. Spiroplasma genic mollicute Spiroplasma kunkelii. Mol. Genet. Genomics 269: diminutum sp. nov., from Culex annulus mosquitoes collected in Tai- 592–602. wan. Int. J. Syst. Bacteriol. 46: 229–233. Zhao, Y., R.W. Hammond, I.M. Lee, B.A. Roe, S. Lin and R.E. Davis. Williamson, D.L., J.R. Adams, R.F. Whitcomb, J.G. Tully, P. Carle, M. 2004a. Cell division gene cluster in Spiroplasma kunkelii: functional Konai, J.M. Bove and R.B. Henegar. 1997. Spiroplasma platyhelix sp. characterization of ftsZ and the first report of ftsA in mollicutes. DNA nov., a new mollicute with unusual morphology and genome size Cell Biol. 23: 127–134. from the dragonfly Pachydiplax longipennis. Int. J. Syst. Bacteriol. 47: Zhao, Y., H. Wang, R.W. Hammond, R. Jomantiene, Q. Liu, S. Lin, B.A. 763–766. Roe and R.E. Davis. 2004b. Predicted ATP-binding cassette systems Williamson, D.L., R.F. Whitcomb, J.G. Tully, G.E. Gasparich, D.L. Rose, in the phytopathogenic mollicute Spiroplasma kunkelii. Mol. Genet. P. Carle, J.M. Bové, K.J. Hackett, J.R. Adams, R.B. Henegar, M. Konai, Genomics 271: 325–338. Family I. Acholeplasmataceae 687

Order III. Acholeplasmatales Freundt, Whitcomb, Barile, Razin and Tully 1984, 348VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.cho.le.plas.ma.ta¢les. N.L. neut. n. Acholeplasma type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Acholeplasmatales the Acholeplasma order.

This order in the class Mollicutes is assigned to a group of wall- Although most mollicutes require exogenous cholesterol or less prokaryotes that do not require sterol for growth and occur serum for growth, all species within the genus Acholeplasma and in a wide variety of habitats, including many vertebrate hosts, some assigned to the genera Asteroleplasma, Spiroplasma, and Meso- insects, and plants. A single family, Acholeplasmataceae, and a sin- plasma do not have that requirement. The species that do not gle genus, Acholeplasma, recognize the prominent and distinct have a sterol requirement can easily be excluded from the sterol- characteristics of the assigned organisms. requiring taxa by tests that measure growth responses to choles- Type genus: Acholeplasma Edward and Freundt 1970, 1AL. terol or to a number of serum-free broth preparations (Edward, 1971; Razin and Tully, 1970; Rose et al., 1993; Tully, 1995). For Further descriptive information instance, the Acholeplasmatales grow through end-point dilutions The trivial name acholeplasma(s) is commonly used when in serum-containing medium and in serum-free preparations, reference is made to species of this order. The initial pro- indicating the absence of a growth requirement for cholesterol. posal for elevation of the acholeplasmas to ordinal rank Analyses of rRNA and other genes have shown that a large ­(Freundt et al., 1984) was based primarily on the universal group of uncultured, plant-pathogenic organisms referred to by lack of a sterol requirement for growth of Acholeplasma the trivial name phytoplasmas (Sears and Kirkpatrick, 1994) are ­species, in addition to other major genetic, nutritional, bio- closely related to acholeplasmas (Lim and Sears, 1992; Toth chemical, and physiological characteristics that distinguish et al., 1994). The 16S rRNA gene sequences for members of the them from other members of the class Mollicutes. A subse- genus Acholeplasma that have been determined so far show that quent proposal for an additional order, Entomoplasmatales the acholeplasmas form two clades, one of which is a sister ­lineage (Tully et al., 1993), within the class to distinguish a group of to the phytoplasmas, although the formal taxonomic assignment mollicutes that are phylogenetically more closely related to of “Candidatus Phytoplasma” proposed gen. nov. (IRPCM Phyto- the Mycoplasmatales than to acholeplasmas necessitated plasma/Spiroplasma Working Team – Phytoplasma Taxonomy ­further revisions within the class. Group, 2004) currently remains incertae sedis.

References Rose, D.L., J.G. Tully, J.M. Bové and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethyl- Edward, D.G. and E.A. Freundt. 1970. Amended nomenclature for ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532. strains related to Mycoplasma laidlawii. J. Gen. Microbiol. 62: 1–2. Sears, B.B. and B.C. Kirkpatrick. 1994. Unveiling the evolutionary rela- Edward, D.G. 1971. Determination of sterol requirement for Mycoplas- tionships of plant pathogenic mycoplasmalike organisms. ASM News matales. J. Gen. Microbiol. 69: 205–210. 60: 307–312. Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Toth, K.F., N. Harrison and B.B. Sears. 1994. Phylogenetic relation- Proposal for elevation of the family Acholeplasmataceae to ordinal ships among members of the class Mollicutes deduced from rps3 gene rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349. sequences. Int. J. Syst. Bacteriol. 44: 119–124. IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy Taxonomy Group. 2004. Description of the genus ‘Candidatus Phy- of the class Mollicutes - proposed elevation of a monophyletic cluster of toplasma’, a taxon for the wall-less non-helical prokaryotes that arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: nov.), with provision for familial rank to separate species with nonhelical 1243–1255. morphology (Entomoplasmataceae fam. nov.) from helical species (Spiro- Lim, P.O. and B.B. Sears. 1992. Evolutionary relationships of a plant- plasmataceae), and emended descriptions of the order Mycoplasmatales, pathogenic mycoplasmalike organism and Acholeplasma laidlawii family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385. deduced from two ribosomal protein gene sequences. J. Bacteriol. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sor- 174: 2606–2611. bitan growth requirements of mollicutes. In Molecular and Diagnos- Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. tic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). J. Bacteriol. 102: 306–310. Academic Press, San Diego, pp. 381–389.

Family I. Acholeplasmataceae Edward and Freundt 1970, 1AL

D a n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.cho.le.plas.ma.ta.ce¢ae. N.L. neut. n. Acholeplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Acholeplasmataceae the Acholeplasma family. Further descriptive information This family is monotypic, so its properties are essentially those Type genus: Acholeplasma Edward and Freundt 1970, 1AL. of the genus Acholeplasma. 688 Family I. Acholeplasmataceae

Genus I. Acholeplasma Edward and Freundt 1970, 1AL

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.cho.le.plas¢ma. Gr. pref. a not; Gr. n. chole bile; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Acholeplasma name intended to indicate that cholesterol, a constituent of bile, is not required. Cells are spherical, with a diameter of about 300 nm, or fila- mentous, 2–5 µm long. Nonmotile. Colonies have a “fried-egg” appearance and may reach 2–3 mm in diameter. Facultatively anaerobic; most strains grow readily in simple media. All mem- bers lack a sterol requirement for growth. Chemo-­organotrophic, most species utilizing glucose and other sugars as the major energy sources. Many strains are capable of fatty acid biosyn- thesis from acetate. Arginine and urea are not hydrolyzed. Pigmented carotenoids occur in some species. All species are resistant, or only slightly susceptible, to 1.5% digitonin. Sapro- phytes found in soil, compost, wastewaters, or commensals of vertebrates, insects, or plants. None are known to be a primary pathogen, but they may cause cytopathic effects in tissue cul- tures. The genome sizes range from about 1500 to 2100 kbp. All species examined utilize the universal genetic code in which UGA is a stop codon. DNA G+C content (mol%): 27–38. Type species: Acholeplasma laidlawii (Sabin 1941) Edward and Freundt 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334). FIGURE 114. Colonies of Acholeplasma laidlawii PG8T (=NCTC 10116T; diameter 0.15–0.25 mm) after 3 d growth on Mycoplasma Experience Further descriptive information Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification = 25×. Image provided by Helena Windsor and David Cells of acholeplasmas typically appear as pleomorphic coc- Windsor. coid, coccobacillary, or short filamentous forms when grown in mycoplasma broth containing 20% horse serum or 1% bovine serum fraction. Viable spherical cells usually have a minimum Most species in the genus are strong fermenters and produce diameter of about 300 nm. Filaments may be as much as 500 nm acid from glucose metabolism, although a few species such as in length, but some longer filaments and branching filaments Acholeplasma parvum may not ferment glucose or other carbohy- occur in some strains. Filaments often show beading with even- drates (Table 143). Fermentation of mannose is usually nega- tual development of coccoid forms. Cellular morphology may tive, although several species do catabolize this carbohydrate. also depend upon the ratio of unsaturated to saturated fatty All Acholeplasma species examined possess a fructose 1,6-diphos- acids in the medium. Adjustment of preparative materials to phate-activated lactate dehydrogenase, which is a property the osmolarity of the culture medium is necessary for proper shared with certain streptococci. morphological examination. Gourlay (1970) found that a fresh isolate of Acholeplasma Most acholeplasmas exhibit heavy turbidity when grown aer- laidlawii from a bovine source was infected with a filamen- obically in broth containing 5–20% serum, usually of horse or tous, single-stranded DNA virus designated L1 (Bruce et al., fetal bovine origin, or when grown in 1% bovine serum frac- 1972; Maniloff, 1992). Later, L2 and L3 viruses were also iso- tion broth at 37°C. Less turbidity is evident when most achole- lated from Acholeplasma laidlawii (Gourlay, 1971, 1972, 1973; plasmas are cultured in serum-free broth and some species may ­Gourlay et al., 1973). L2 virus is a quasi-spherical, double- be inhibited in media containing 20% horse serum. Strains of stranded DNA virus (Maniloff et al., 1977), and L3 is a short- some acholeplasmas (Acholeplasma morum, Acholeplasma modi- tailed phage with double-stranded DNA (Garwes et al., 1975; cum, and Acholeplasma axanthum) may not grow well in serum- Gourlay, 1974; Haberer et al., 1979; Maniloff et al., 1977). free medium unless glucose and some fatty acids (Tween 80 and Another virus isolated from Acholeplasma laidlawii is L172, a palmitic acid) are included. Colonies on solid medium contain- single-stranded DNA, quasi-spherical virus that is different ing serum or bovine serum fraction are usually large (100– from L1 (Liska, 1972). Two viruses have been isolated from 200 nm in diameter) with the classical “fried-egg” appearance other acholeplasmas, including one from Acholeplasma modi- after 24–72 h at 37°C (Figure 114). Colonies of Acholeplasma cum, designated M1 (Congdon et al., 1979), and from Achole- axanthum and several other acholeplasmas may show only cen- plasma oculi strain PG49 (designated O1) (Ichimaru and tral zones of growth into the agar or other unusual colony forms, Nakamura, 1983). The nucleic acid structure of the last two such as mulberry-like colonies. Most acholeplasmas display viruses has not been defined. optimum growth at 37°C. Growth is much slower at 25–27°C Antisera to filter-cloned whole-cell antigens are utilized in sev- and strains may require 7–10 d to reach the turbidity observed eral serological techniques to assess the antigenic structure of after 24 h at 37°C. Species of plant origin (Acholeplasma brassicae acholeplasmas and to provide identification of the organism to and Acholeplasma palmae) have an optimum growth temperature the species level (Tully, 1979). The three most useful ­techniques of 30°C. are growth inhibition (Clyde, 1983), plate ­immunofluorescence Genus I. Acholeplasma 689

TABLE 143. Differential characteristics of the species of the genus Acholeplasma a

Characteristic A. laidlawii A. axanthum A. brassicae cavigenitalium A. A. equifetale A. granularum A. hippikon A. modicum A. morum A. multilocale A. oculi A. palmae A. parvum A. pleciae A. vituli Glucose fermentation + + + + + + + + + + + + − + + Mannose fermentation − − − − + − + − − + − − nd nd + Arbutin hydrolysis + + − − nd − nd − + nd + − nd nd − Esculin hydrolysis + + nd nd nd − nd − + nd + nd − nd − Film and spots + − nd − + − + − − + − nd nd nd − Benzyl viologen + + + + + + + + + − + + + nd nd reduction DNA G+C content 31–36 31 35.5 36 30.5 30–32 33 29 34 31 27 30 29 31.6 37.6–38.3 (mol%) aSymbols: +, >85% positive; −, 0–15% positive; nd, not determined.

(Gardella et al., 1983; Tully, 1973), and metabolism inhibition species (Brown et al., 2007). Techniques for isolation of achole- (Taylor-Robinson, 1983). plasmas from animal tissues and from cell cultures have been Acholeplasmas may be the most common mollicutes in ver- described (Tully, 1983). Although colonies are occasionally tebrate animals and they are found frequently in the upper first detected on blood agar, complex undefined media such respiratory tract and urogenital tract of such hosts (Tully, 1979, as American Type Culture Collection medium 988 (SP-4) are 1996). Eukaryotic cells in continuous culture are frequently con- usually required for primary isolation and maintenance. Cell taminated with acholeplasmas, primarily from the occurrence of wall-targeting antibiotics are included to discourage growth of acholeplasmas in animal serum used in tissue culture media. At other bacteria. Phenol red facilitates detection of species that least five Acholeplasma species have been identified on plant sur- excrete acidic or alkaline metabolites. Commonly used alterna- faces (Acholeplasma axanthum, Acholeplasma brassicae, Acholeplasma tives such as Frey’s, Hayflick’s and Friis’ media differ from SP-4 laidlawii, Acholeplasma oculi, and Acholeplasma palmae), possibly mainly in the proportions of inorganic salts, amino acids, serum representing contamination from insects. However, with the sources, and types of antibiotics included. Broths are incubated exception of Acholeplasma pleciae (Knight, 2004), the only achole- aerobically at 37°C for 14 d and examined periodically for tur- plasmas identified from insects have been from mosquitoes. bidity or pH changes, either to acid or alkaline levels. Tubes Acholeplasma laidlawii was identified in a pool of Anopheles sinensis, showing turbidity are plated to agar prepared from the same and a strain of Acholeplasma morum was present in a pool of Armig- medium formulation, and the plates are incubated at 37°C in eres subalbatus (D.L. Williamson and J.G. Tully, unpublished). an atmosphere of 95% N2, 5% CO2, as in the GasPak system. Little evidence exists for a pathogenic role of acholeplasmas in Tubes without obvious turbidity should be plated at the end of natural diseases. The widespread distribution of acholeplasmas the 14-d incubation period. Initial isolates may contain a mix- in both healthy and diseased animal tissues and of antibodies ture of species, so cloning by repeated filtration through mem- against acholeplasmas in most animal sera complicates experi- brane filters with a pore size of 450 or 220 nm is essential. The mental pathogenicity studies. However, Acholeplasma axanthum initial filtrate and dilutions of it are cultured on solid medium was pathogenic for goslings and young goose embryos (Kisary and an isolated colony is subsequently picked from a plate on et al., 1975, 1976). Inoculation into leafhoppers, ­including which only a few colonies have developed. This colony is used those known to be vectors of plant mycoplasma diseases, shows to found a new cultural line, which is then expanded, filtered, multiplication and prolonged persistence of acholeplasmas in plated, and picked two additional times. Identification is con- host tissues (Eden-Green and Markham, 1987; Whitcomb et al., firmed by additional biochemical and serological tests. 1973; Whitcomb and Williamson, 1975), but there is no evi- dence that the few Acholeplasma species found on plant surfaces Maintenance procedures play any role in plant or insect disease. Stock acholeplasma cultures can be maintained in either A few recent reports are available on the antibiotic sensitivity mycoplasma broth medium containing 5–20% serum or in of acholeplasmas and whether the actions of these drugs are the serum-fraction broth formulation at room temperature inhibitory to growth or kill cells. Acholeplasmas are sensitive (25–30°C) with only weekly transfer (Tully, 1995). Maintenance to the following antibiotics (minimum inhibitory concentration is best in broth medium devoid of glucose, since excess acid range in µg/ml): tetracycline, 0.5–25.0; erythromycin, 0.03–1.0; production reduces viability. Stock cultures can also be main- lincomycin, 0.25–1.0; tylosin tartarate, 0.1–12.5; and kanamy- tained indefinitely when frozen at −70°C. Agar colonies can cin, 20–200 (Kato et al., 1972; Lewis and Poland, 1978; Ogata also be maintained for 1–2 weeks at 25°C if plates are sealed et al., 1971). to prevent drying. For optimum preservation, acholeplasmas should be lyophilized directly in the culture medium when the Enrichment and isolation procedures broth cultures reach a mid-exponential phase, usually 1–2 d at Typical steps in isolation of all mollicutes were outlined in the 37°C. Lyophilized cultures should be sealed under vacuum and recently revised minimal standards for descriptions of novel stored at 4°C (Leach, 1983). 690 Family I. Acholeplasmataceae

Differentiation of the genus Acholeplasma from each other and some heterogeneity occurred among six from other genera different Acholeplasma oculi strains. Mesoplasma pleciae was first isolated from the hemolymph of Properties that partially fulfill criteria for assignment to the a larva of a Plecia corn root maggot and assigned to the genus class Mollicutes (Brown et al., 2007) include absence of a cell Mesoplasma because sustained growth occurred in serum-free wall, filterability, and the presence of conserved 16S rRNA mycoplasma broth only when the medium contained 0.04% gene sequences. They usually possess two 16S rRNA operons. Tween 80 fatty acid mixture (Tully et al., 1994a). However, 16S ­Aerobic or facultative anaerobic growth in artificial media rRNA gene sequence similarities and its preferred use of UGG and the absence of a requirement for sterols or cholesterol rather than UGA to encode tryptophan support proper reclas- for growth exclude assignment to the genera Anaeroplasma, sification as Acholeplasma pleciae comb. nov. (Knight, 2004); the Asteroleplasma, “Candidatus Phytoplasma”, Mycoplasma, or Ure- type strain is PS-1T (Tully et al., 1994a). aplasma. Absence of a spiral cellular morphology, regular asso- Mycoplasma feliminutum was first described during a time ciation with a vertebrate host or fluids of vertebrate origin, and when the only named genus of mollicutes was Mycoplasma. regular use of the codon UGG to encode tryptophan (Knight, Its publication coincided with the first proposal of the genus 2004) and UGA as a stop codon (Tanaka et al., 1989, 1991) Acholeplasma (Edward and Freundt, 1969, 1970), with which support exclusion from the genera Spiroplasma, Entomoplasma, Mycoplasma feliminutum is properly affiliated through estab- or Mesoplasma. Reduction of the redox indicator benzyl violo- lished phenotypic (Heyward et al., 1969) and 16S rRNA gene gen has been reported to be fairly specific for differentiation sequence (Brown et al., 1995; Johansson and Pettersson, 2002) of the genus Acholeplasma from other mollicutes (Pollack et al., similarities. This explains the apparent inconsistencies with its 1996a). Only Acholeplasma multilocale failed to give a positive assignment to the genus Mycoplasma. The name Mycoplasma reaction, although several Mesoplasma and Entomoplasma spe- feliminutum should therefore be revised to Acholeplasma felim- cies yielded variable responses to the test (Pollack et al., 1996a). inutum comb. nov.; the type strain is BenT (=ATCC 25749T; Hey- Most acholeplasmas have membrane-localized NADH oxidase ward et al., 1969). activity, in comparison to the NADH oxidase activity located The lack of signature enzymic activities cast serious doubt in the cytoplasm of other genera within the class. Another on the status of Acholeplasma multilocale PN525T as an authentic special characteristic is the occurrence in most acholeplasmas member of the genus Acholeplasma (Pollack et al., 1996b). It of unique pyrimidine enzymic activities, especially a dUTPase may be affiliated with an unrecognized metabolic subgroup, enzyme, with the possible exception again of Acholeplasma mul- but it seems more likely to be a strain of Mycoplasma or Ento- tilocale (Pollack et al., 1996b). Acholeplasmas may possess a moplasma. number of other biological characteristics that may distinguish them from other genera within the class Mollicutes, including Acknowledgements polyterpenol synthesis (Smith and Langworthy, 1979), posi- tional distribution of fatty acids (Rottem and Markowitz, 1979), The major contributions to the foundation of this material by the presence of superoxide dismutase (Kirby et al., 1980; Lee Joseph G. Tully are gratefully acknowledged. and Kenny, 1984; Lynch and Cole, 1980; O’Brien et al., 1981), Further reading and the presence of spacer tRNA (­Nakagawa et al., 1992). However, most of these features have not been established for Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ure- even a majority of Acholeplasma species. aplasmas, spiroplasmas, and related organisms. In Topley and Wilson, Principles and Practice of Microbiology, 9th edn, vol. 2 (edited by Balows and Duerden). Arnold Publishers, Taxonomic comments London, pp. 799–827. Acholeplasma genome sizes range from 1215 to 2095 kbp by Tully, J.G. 1989. Class Mollicutes: new perspectives from plant pulsed-field gel electrophoresis or complete DNA sequencing, and arthropod studies. In The Mycoplasmas (edited by Whit- but most are in a more narrow range of 1215–1610 kbp (Carle comb and Tully). Academic Press, San Diego, pp. 1–31. et al., 1993; Neimark et al., 1992) that overlaps with genome Differentiation of the species of the genus sizes of many Spiroplasma species. Tests of eight Acholeplasma Acholeplasma species showed less than 8% DNA–DNA hybridization between type strains and surprisingly extensive genomic heterogene- Esculin hydrolysis by a b-d-glucosidase and arbutin hydrolysis ity within species (Aulakh et al., 1983; Stephens et al., 1983a, are sometimes useful diagnostic tests for differentiation of b). The highest level of relatedness, 21% DNA–DNA hybrid- some acholeplasmas (Bradbury, 1977; Rose and Tully, 1983). ization, was between the type strains of Acholeplasma laidlawii The production of carotenoid pigments, principally neuro- and Acholeplasma granularum. Some strain pairs, such as within sporene, has been used to differentiate some acholeplasmas, Acholeplasma laidlawii, shared as little as 40% DNA–DNA hybrid- especially Acholeplasma axanthum and Acholeplasma modicum ization, differences that in other genera would have justified (Mayberry et al., 1974; Smith and Langworthy, 1979; Tully subdivision of an apparently diverse strain complex into com- and Razin, 1970). Carotenoids are also synthesized in some ponent species. However, no polyphasic taxonomic basis was strains of Acholeplasma laidlawii under certain growth condi- found to support such designations. Restriction endonuclease tions (Johansson, 1974). The “film and spots” reaction, which digest patterns also reflect heterogeneity within some species occurs in a number of Mycoplasma and several Acholeplasma spe- (Razin et al., 1983). The DNA–DNA hybridization and restric- cies, relates to the production of crystallized calcium soaps of tion digest patterns of eight Acholeplasma axanthum strains iso- fatty acids on the surface of agar plates (Edward, 1954; ­Fabricant lated from a variety of hosts and habitats differed markedly and Freundt, 1967). Fatty acids are liberated from the serum or Genus I. Acholeplasma 691 supplemental egg yolk (Fabricant and Freundt, 1967; Thorns nary differentiation can be by PCR and DNA sequencing using and Boughton, 1978) in the agar medium by the lipolytic activ- primers specific for bacterial 16S rRNA genes or the 16S–23S ity of the organisms. Failure to cross-react with antisera against intergenic region. A similarity matrix relating the candidate previously recognized species provides evidence for species strain to its closest neighbors, usually species with >0.94 16S novelty. For this reason, deposition of antiserum against a rRNA gene sequence similarity, will suggest an assemblage of novel type strain into a recognized collection is still manda- related species that should be examined for serological cross- tory for new species descriptions (Brown et al., 2007). Prelimi- reactivities.

List of species of the genus Acholeplasma

1. Acholeplasma laidlawii (Sabin 1941) Edward and Freundt growth around their center. Agar colonies produce zones 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334) of b-hemolysis by the overlay technique. Growth in media devoid of serum or serum fraction is much poorer than for ¢ laid.law i.i. N.L. gen. masc. n. laidlawii of Laidlaw, named other acholeplasmas. Minimal nutritional requirements are after Patrick P. Laidlaw, one of the microbiologists who first poorly defined, but marked stimulation of growth with poly- isolated this species. oxyethylene sorbitan (Tween 80) suggests a requirement This is the type species of the genus Acholeplasma. Fila- for fatty acids. Temperature range for growth is 22–37°C ments, usually relatively short, although much longer with optimum growth at 37°C. Synthesis of carotenoid pig- branched filaments may develop in media with certain ments can be demonstrated only when large volume cul- ratios of saturated to unsaturated fatty acids. Coccoid forms tures are tested. Produces sphingolipids. No evidence for may predominate in certain cultures including co-culture pathogenicity. with eukaryotic cells. Agar colonies are large for a mollicute Source: originally isolated from murine leukemia tissue cul- and exhibit well-developed central zones and peripheral ture cells, but numerous subsequent isolations of the organ- growth on horse serum agar. On serum-free agar, colonies ism from bovine serum and a variety of bovine tissue sites are smaller and may show only the central zone of growth (nasal cavity, lymph nodes, kidney) suggest cell-culture con- into the agar. Relatively strong turbidity is produced during tamination was of bovine serum origin. Also isolated from growth in broth containing serum. Temperature range for variety of other animals and surfaces of some plants. growth is 20–41°C with optimum at 37°C, even for strains DNA G+C content (mol%): 31 (Bd). recovered from plant or non-animal sources. Usually pro- Type strain: S-743, ATCC 25176, NCTC 10138. duces large amounts of carotenoids when cultivated in the Sequence accession no. (16S rRNA gene): AF412968. presence of PPLO serum fraction (Difco). 3. Acholeplasma brassicae Tully, Whitcomb, Rose, Bové, Carle, Serologically distinct from most established species in Somerson, Williamson and Eden-Green 1994b, 683VP the genus, but partial cross-reactions may occur with Achole- plasma granularum strains. DNA–DNA hybridization between bras.si¢cae. L. fem. gen. n. brassicae of cabbage, referring to strains of this species range from 40 to >80%. Acholeplasma the plant origin of the organism. granularum strain BTS-39T showed 20% hybridization with Cells are primarily coccoid. Temperature range for Acholeplasma laidlawii strain PG8T. Pathogenicity has not growth is 18–37°C. Optimal growth occurs at 30°C. No evi- been established. dence for pathogenicity. Source: isolated from sewage, manure, humus, soil, and Source: isolated as a surface contaminant from broccoli many animal hosts and their tissues, including some isolates (Brassica oleracea var. italica). from the human oral cavity, vagina, and wounds. Has been DNA G+C content (mol%): 35.5 (Bd, Tm, HPLC). recovered from the surfaces of some plants, although few Type strain: 0502, ATCC 49388. isolations have been reported from insect hosts. Frequent Sequence accession no. (16S rRNA gene): AY538163. contaminant of eukaryotic cell cultures. 4. Acholeplasma cavigenitalium Hill 1992, 591VP DNA G+C content (mol%): 31.7–35.7 (Bd, Tm). Type strain: ATCC 23206, PG8, NCTC 10116, CIP 75.27, ca.vi.ge.ni.ta¢li.um. N.L. n. cavia guinea pig (Cavia cobaya); NBRC 14400. L. pl. n. genitalia -ium the genitals; N.L. pl. gen. n. cavigenita- Sequence accession no. (16S rRNA gene): U14905. lium of guinea pig genitals. Further comment: on the Approved Lists of Bacterial Names Pleomorphic cells, mostly coccoid. Grows on broth or and on the Approved Lists of Bacterial Names (Amended agar medium under aerobic conditions, with optimum tem- Edition), this taxon is incorrectly cited as Acholeplasma laid- perature between 35 and 37°C. Colonies on agar medium lawii [Freundt 1955 (sic)] Edward and Freundt (1970). have typical fried-egg appearance. Originally described as a 2. Acholeplasma axanthum Tully and Razin 1970, 754AL non-fermenter, but the type strain ferments glucose. Does not grow well on SP-4 broth or in horse serum broth, but a.xan¢thum. Gr. pref. a not, without; Gr. adj. xanthos -ê -on grows well on simple base medium with additions of 10–15% yellow; N.L. neut. adj. axanthum without yellow (pigment). fetal bovine serum. No evidence for pathogenicity. Predominantly coccobacillary and coccoid with a few Source: isolated from the vagina of guinea pigs. short myceloid elements. Large colonies with clearly DNA G+C content (mol%): 36 (Bd). marked centers form on horse serum agar; colonies on Type strain: GP3, NCTC 11727, ATCC 49901. serum-free agar are smaller and usually lack the peripheral Sequence accession no. (16S rRNA gene): AY538164. 692 Family I. Acholeplasmataceae

5. Acholeplasma entomophilum Tully, Rose, Carle, Bové, Further comment: with the proposal of the order Ento- Hackett and Whitcomb 1988, 166VP moplasmatales (Tully et al., 1993), Acholeplasma florum was en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. phi- transferred to the family Entomoplasmataceae. The name lum (from Gr. neut. adj. philon) friend, loving; N.L. neut. Acholeplasma florum was therefore revised to Mesoplasma adj. entomophilum insect-loving. florum comb. nov. The type strain is L1T (=ATCC 33453T; Cells are pleomorphic, but primarily coccoid. Colonies McCoy et al., 1984). on solid medium usually have a fried-egg appearance. Acid 8. Acholeplasma granularum (Switzer 1964) Edward and Fre- is produced from glucose, but not mannose. Carotenoids undt 1970, 2AL (Mycoplasma granularum Switzer 1964, 504) are not produced. “Film and spot” reaction is negative. gra.nu.la¢rum. N.L. fem. n. granula (from L. neut. n. granu- Agar colonies hemadsorb guinea pig erythrocytes. Strains lum) a small grain, a granule; N.L. gen. pl. n. granularum of require 0.4% Tween 80 or fatty acid supplements for growth small grains, made up of granules, granular. in serum-free media. Temperature range for growth is 23–32°C, with optimum growth at about 30°C. Pathogenic- Cells are pleomorphic, with short filaments and coc- ity has not been established. coid cells. Colonies on solid medium are large with clearly Source: isolated from gut contents of tabanid flies, beetles, marked central zones and a fried-egg appearance. Colonies butterflies, honey bees, and moths, and from flowers. on serum-free medium are smaller and may lack the periph- DNA G+C content (mol%): 30 (Bd). eral zone of growth around central core. Temperature Type strain: TAC, ATCC 43706. range for growth is 22–37°C, with optimum around 37°C. Sequence accession no. (16S rRNA gene): M23931. Agar colonies produce a zone of b-hemolysis by the overlay Further comment: with the proposal of the order Ento- technique using sheep erythrocytes. DNA–DNA hybridiza- moplasmatales (Tully et al., 1993), Acholeplasma entomophi- tion studies showed 20–22% hybridization with Acholeplasma lum was transferred to the family Entomoplasmataceae. The laidlawii, but none with other acholeplasmas. Pathogenic- name Acholeplasma entomophilum was therefore revised to ity has not been established. Aerosol challenge of specific Mesoplasma entomophilum comb. nov. The type strain is TACT pathogen-free pigs did not induce clinical or histological (=ATCC 43706T; Tully et al., 1988). evidence of disease. Source: isolated frequently from the nasal cavity of swine, AL 6. Acholeplasma equifetale Kirchhoff 1978, 81 with occasional isolates from swine lung and feces. Also eq.ui.fe.ta¢le. L. n. equus horse; N.L. adj. fetalis -is -e pertain- isolated from the conjunctivae and nasopharynx of horses, ing to the fetus; N.L. neut. adj. equifetale pertaining to the and the genital tract of guinea pigs. Occasional contami- horse fetus. nant of eukaryotic cell cultures.

Cells are pleomorphic, but predominantly coccoid. Col- DNA G+C content (mol%): 30.5–32.4 (Tm, Bd). onies on solid medium containing serum usually have a Type strain: BTS-39, ATCC 19168, NCTC 10128. fried-egg appearance; on serum-free medium, colonies are Sequence accession no. (16S rRNA gene): AY538166. similar, but usually smaller. Growth temperature range is 9. Acholeplasma hippikon Kirchhoff 1978, 81AL 22–37°C. Pathogenicity has not been established. hip.pi¢kon. Gr. neut. adj. hippikon pertaining to the horse. Source: isolated from the lung and liver of aborted horse fetuses. Also recovered from the respiratory tract of appar- Cells are pleomorphic with predominantly coccoid ently normal horses and the respiratory tract and cloacae of forms. Colonies on solid medium containing horse serum broiler chickens (Bradbury, 1978). typically have a fried-egg appearance, with smaller colonies DNA G+C content (mol%): 30.5 (Bd). on serum-free agar medium. Growth occurs over a tempera- Type strain: C112, ATCC 29724, NCTC 10171. ture range of 22–37°C, with optimal growth at 35–37°C. Agar Sequence accession no. (16S rRNA gene): AY538165. colonies produce b-hemolysis with the overlay technique, Further comment: Kirchhoff is incorrectly cited as “Kirchoff”­ using a variety of animal red blood cells. ­Pathogenicity has on the Approved Lists of Bacterial Names. not been established. Source: isolated from the lung of aborted horse fetuses. 7. Acholeplasma florum McCoy, Basham, Tully, Rose, Carle DNA G+C content (mol%): 33.1 (Bd). VP and Bové 1984, 14 Type strain: C1, ATCC 29725, NCTC 10172. flo¢rum. L. gen. p1. n. florum of flowers, indicating the Sequence accession no. (16S rRNA gene): AY538167. recovery site of the organism. Further comment: Kirchhoff is incorrectly cited as “Kirchoff”­ Cells are ovoid. Colonies on agar are umbonate. Films on the Approved Lists of Bacterial Names. and spots are produced on serum-containing media. Glu- 10. Acholeplasma modicum Leach 1973, 147AL cose is utilized, but mannose is not. Carotenes are not pro- duced, nor is b-d-glucosidase. Pathogenicity has not been mo¢di.cum. L. neut. adj. modicum moderate, referring to established. moderate growth. Source: the known strains were isolated from flower sur- Cells are pleomorphic, with spherical, ring-shaped, and faces. coccobacillary forms. Colonies on solid medium are dis- DNA G+C content (mol%): 27.3 (Bd). tinctly smaller than those of most other acholeplasmas. Type strain: L1, ATCC 33453. Very small colonies without peripheral zones of growth are Sequence accession nos: AF300327 (16S rRNA gene), noted on serum-free solid medium. Very light turbidity is NC_006055 (strain L1T complete genome). observed in serum-free broth, but more turbidity is found Genus I. Acholeplasma 693

in broth containing serum. Growth temperature range is Cells are pleomorphic, including spherical, ring-shaped, 22–37°C, with optimum growth around 35–37°C. Can be and coccobacillary forms. Medium-sized colonies with typi- shown to produce carotenoids when large volumes of cells cal fried-egg appearance are formed on horse serum agar. are examined. Agar colonies produce a- or b-hemolysis by Colonies on serum-free agar are smaller and may lack the the overlay technique using sheep, ox, or guinea pig red peripheral growth around the central core. Growth occurs blood cells. Pathogenicity has not been established. at temperatures of 25–37°C. Agar colonies produce zones of Source: isolated from various tissues of cattle, including hemolysis by the overlay technique using sheep red blood blood, bronchial lymph nodes, thoracic fluids, lungs, and cells. semen. Also isolated from nasal secretions of pigs, and occa- Pathogenicity is not well established. Intravenous inoc- sionally from chickens, turkeys, and ducks. ulation of goats produced signs of pneumonia and death

DNA G+C content (mol%): 29.3 (Tm). within 6 d. Conjunctival inoculation of goats produced mild Type strain: PG49, ATCC 29102, NCTC 10134. conjunctivitis. Sequence accession no. (16S rRNA gene): M23933. Source: isolated from the conjunctiva of goats with kera- toconjunctivitis; porcine nasal secretions; equine nasophar- 11. Acholeplasma morum Rose, Tully and Del Giudice 1980, ynx, lung, spinal fluid, joint, and semen; the urogenital 653VP tract of cattle; and the external genitalia of guinea pigs. mor¢um. L. n. morum a mulberry, denoting the mulberry- Present in amniotic fluid of pregnant women (Waites et al., like appearance of agar colonies of the organism. 1987). Occasionally isolated from ducks and turkeys, with Cells are pleomorphic, predominantly coccoid or cocco- unreported isolations from an ostrich. Also several isola- bacillary forms, but with some beaded filaments. Colonies tions from palm trees and other plants (Eden-Green and on solid medium without serum supplements are very small Tully, 1979; Somerson et al., 1982). Isolations from eukary- in size and have only central zones without any peripheral otic cell cultures may represent contamination of bovine growth. Optimal growth on solid medium occurs with a origin. 10% serum concentration and colony growth appears to be DNA G+C content (mol%): 27 (Tm). suppressed in a medium with 20% serum. Optimal growth Type strain: 19-L, ATCC 27350, NCTC 10150. in broth is apparent when 5–10% serum is added or when Sequence accession no. (16S rRNA gene): U14904. 1% bovine serum fraction supplements are added, but Further comment: originally named Acholeplasma oculusi poor growth occurs in broth containing 20% horse serum. by al-Aubaidi et al. (1973); the orthographic error was cor- Growth in serum-free broth usually requires some fatty acid rected by al-Aubaidi (1975). supplements, such as palmitic acid or polyoxyethylene sorbi- tan (Tween 80). Temperature range for growth is 23–37°C, 14. Acholeplasma palmae Tully, Whitcomb, Rose, Bové, Carle, VP with optimum growth at about 35–37°C. Somerson, Williamson and Eden-Green 1994b, 683 Pathogenicity has not been established. Calf kidney cell cul- pal¢mae. L. fem. gen. n. palmae of a palm tree, referring to tures containing the organism show cytopathogenic effects. the plant from which the organism was isolated. Source: originally recovered from commercial fetal bovine Cells are primarily coccoid. Colonies on solid medium serum and from calf kidney cultures containing fetal bovine usually have a fried-egg appearance. The temperature serum. One isolation, in broth containing horse serum, was range for growth is 18–37°C, with optimal growth occurring from a pool of Armigeres subalbatus mosquitoes collected by at 30°C. No evidence for pathogenicity. It is one of the clos- Leon Rosen in Taiwan in 1978 (strain SP7; D.L. Williamson est phylogenetic relatives of the phytoplasmas. and J.G. Tully, unpublished). Source: isolated from the crown tissues of a palm tree DNA G+C content (mol%): 34.0 (T ). m (Cocos nucifera) with lethal yellowing disease. Type strain: 72-043, ATCC 33211, NCTC 10188. DNA G+C content (mol%): 30 (Bd, Tm, HPLC). Sequence accession no. (16S rRNA gene): AY538168. Type strain: J233, ATCC 49389. 12. Acholeplasma multilocale Hill, Polak-Vogelzang and ­Angulo Sequence accession no. (16S rRNA gene): L33734. VP 1992, 516 15. Acholeplasma parvum Atobe, Watabe and Ogata 1983, 348VP mul.ti.lo.ca¢le. L. adj. multus many, numerous; L. adj. localis par¢vum. L. neut. adj. parvum small, intended to refer to -is -e of or belonging to a place, local; N.L. neut. adj. multilo- the poor biochemical activities and tiny agar colonies of the cale referring to more than one location. organism. Pleomorphic cells. Colonies on agar medium have a typical fried-egg appearance. Organisms grow well in broth Pleomorphic coccobacillary cells. Colonies on agar medium at 35–37°C. No evidence for pathogenicity. medium present a typical fried-egg appearance under both Source: isolated from the nasopharynx of a horse and the aerobic and anaerobic conditions. Initial reports of growth feces of a rabbit. in the absence of cholesterol or serum have been made, but DNA G+C content (mol%): 31 (Bd). growth on serum-free medium is not well confirmed. The Type strain: PN525, NCTC 11723, ATCC 49900. organism does not grow in most standard media for achole- Sequence accession no. (16S rRNA gene): AY538169. plasmas or in most other medium formulations for sterol- requiring mycoplasmas. Needs special growth factor of 1% 13. Acholeplasma oculi corrig. al-Aubaidi, Dardiri, Muscoplatt phytone or soytone peptone supplements; growth is some- AL and McCauley 1973, 126 times better with the addition of 15% fetal bovine serum. o¢cu.li. L. n. oculus the eye; L. gen. n. oculi of the eye. Organisms grow on agar better than in broth; growth is 694 Family I. Acholeplasmataceae

­better under aerobic conditions than under anaerobic pioneering studies of sterol-nonrequiring mollicutes that ­conditions and better at 22–30°C than at 37°C. No evidence occur in soil and compost. of fermentation of any carbohydrate, including glucose, Cells are primarily coccoid. Colonies on solid medium salicin, and esculin. No evidence for pathogenicity. ­usually have the appearance of fried-eggs. Acid produced Source: isolated from the oral cavities and vagina of from glucose and mannose. Colonies on agar hemadsorb healthy horses. guinea pig erythrocytes. Temperature range for growth is DNA G+C content (mol%): 29.1 (Tm). 20–35°C; optimum growth occurs at 28°C. No evidence for Type strain: H23M, ATCC 29892, NCTC 10198. pathogenicity. Sequence accession no. (16S rRNA gene): AY538170. Source: isolated from floral surfaces of a sweet orange 16. Acholeplasma pleciae (Tully, Whitcomb, Hackett, Rose, (Citrus sinensis) and wild angelica (Angelica sylvestris). ­Henegar, Bové, Carle, Williamson and Clark 1994a) Knight DNA G+C content (mol%): 30 (Bd). 2004, 1952VP (Mesoplasma pleciae Tully, Whitcomb, Hackett, Rose, Type strain: F7, ATCC 49495. Henegar, Bové, Carle, Williamson and Clark 1994a, 690) Sequence accession no. (16S rRNA gene): AY351331. Further comment: with the proposal of the order Ento- ple.ci¢ae. N.L. gen. n. pleciae of Plecia, referring to the genus moplasmatales (Tully et al., 1993), Acholeplasma seiffertii was of corn maggot (Plecia sp.) from which the organism was transferred to the family Entomoplasmataceae. The name first isolated. Acholeplasma seiffertii was therefore revised to Mesoplasma Cells are primarily coccoid. Colonies on solid media seiffertii comb. nov. The type strain is F7T (=ATCC 49495T; incubated under anaerobic conditions at 30°C have a fried- Bonnet et al., 1991). egg appearance. Supplements of 0.04% polyoxyethylene 18. Acholeplasma vituli Angulo, Reijgers, Brugman, Kroesen, sorbitan (Tween 80) are required for growth in serum-free Hekkens, Carle, Bové, Tully, Hill, Schouls, Schot, Roholl media. Temperature range for growth is 18–32°C, with and Polak-Vogelzang 2000, 1130VP optimal growth at 30°C. Agar colonies do not hemadsorb guinea pig erythrocytes. No evidence for pathogenicity. vi.tu¢li. L. n. vitulus calf; L. gen. n. vituli of calf, referring to the Source: originally isolated from the hemolymph of a larva provenance or occurrence of the organism in fetal calf serum. of the corn root maggot (Plecia sp.). Cells are predominantly coccoid in shape. Colonies on

DNA G+C content (mol%): 31.6 (Bd, Tm, HPLC). solid media demonstrate a fried-egg appearance under Type strain: PS-1, ATCC 49582. both aerobic and anaerobic conditions. Temperature range Sequence accession no. (16S rRNA gene): AY257485. for growth is 25–37°C. No evidence for pathogenicity. 17. Acholeplasma seiffertii Bonnet, Saillard, Vignault, Garnier, Source: isolated from fetal bovine serum or contaminated Carle, Bové, Rose, Tully and Whitcomb 1991, 48VP eukaryotic cell cultures containing serum. DNA G+C content (mol%): 38.3 (Bd), 37.6 (Tm). seif.fer¢ti.i. N.L. gen. masc. n. seiffertii of Seiffert, in honor Type strain: FC 097-2, ATCC 700667, CIP 107001. of Gustav Seiffert, a German microbiologist who performed Sequence accession no. (16S rRNA gene): AF031479.

References Brown, D., G. McLaughlin and M. Brown. 1995. Taxonomy of the feline al-Aubaidi, J.M. 1975. Orthographic errors in the name Acholeplasma mycoplasmas Mycoplasma felifaucium, Mycoplasma feliminutum, Myco- oculusi. Int. J. Syst. Bacteriol. 25: 221. plasma felis, Mycoplasma gateae, Mycoplasma leocaptivus, Mycoplasma al-Aubaidi, J.M., A.H. Dardiri, C.C. Muscoplatt and E.H. McCauley. leopharyngis, and Mycoplasma simbae by 16S rRNA gene sequence com- 1973. Identification and characterization of Acholeplasma oculusi spec. parisons. Int. J. Syst. Bacteriol. 45: 560–564. nov. from the eyes of goats with keratoconjunctivitis. Cornell Vet. 63: Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal stan- 117–129. dards for description of new species of the class Mollicutes (division Angulo, A.F., R. Reijgers, J. Brugman, I. Kroesen, F.E.N. Hekkens, Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. P. Carle, J.M. Bové, J.G. Tully, A.C. Hill, L.M. Schouls, C.S. Schot, Bruce, J., R.N. Gourlay, R. Hull and D.J. Garwes. 1972. Ultrastructure of P.J.M. Roholl and A.A. Polak-Vogelzang. 2000. Acholeplasma vituli sp. Mycoplasmatales virus laidlawii I. J. Gen. Virol. 16: 215–221. nov., from bovine serum and cell cultures. Int. J. Syst. Evol. Micro- Carle, P., D.L. Rose, J.G. Tully and J.M. Bové. 1993. The genome size of biol. 50: 1125–1131. spiroplasmas and other mollicutes. Int. Org. Mycoplasmol. Lett. 2: 263. Atobe, H., J. Watabe and M. Ogata. 1983. Acholeplasma parvum, a new Clyde, W.A., Jr. 1983. Growth inhibition tests. In Methods in Mycoplas- species from horses. Int. J. Syst. Bacteriol. 33: 344–349. mology, vol. 1 (edited by Razin and Tully). Academic Press, New Aulakh, G.S., E.B. Stephens, D.L. Rose, J.G. Tully and M.F. Barile. 1983. York, pp. 405–410. Nucleic acid relationships among Acholeplasma species. J. Bacteriol. Congdon, A.L., E.S. Boatman and G.E. Kenny. 1979. Mycoplasmatales 153: 1338–1341. virus MV-M1: discovery in Acholeplasma modicum and preliminary Bonnet, F., C. Saillard, J.C. Vignault, M. Garnier, P. Carle, J.M. Bové, characterization. Curr. Microbiol. 3: 111–115. D.L. Rose, J.G. Tully and R.F. Whitcomb. 1991. Acholeplasma seiffertii sp. Eden-Green, S.J. and P.G. Markham. 1987. Multiplication and persis- nov., a mollicute from plant surfaces. Int. J. Syst. Bacteriol. 41: 45–49. tence of Acholeplasma spp. in leafhoppers. J. Invertebr. Pathol. 49: Bradbury, J. 1977. Rapid biochemical tests for characterization of the 235–241. Mycoplasmatales. J. Clin. Microbiol. 5: 531–534. Eden-Green, S.J. and J.G. Tully. 1979. Isolation of Acholeplasma spp. Bradbury, J.M. 1978. Acholeplasma equifetale in broiler chickens. Vet. Rec. from coconut palms affected by lethal yellowing disease in Jamaica. 102: 516. Curr. Microbiol. 2: 311–316. Genus I. Acholeplasma 695

Edward, D.G. 1954. The pleuropneumonia group of organisms: a Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Achole- review, together with some new observations. J. Gen. Microbiol. 10: plasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene 27–64. sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952. Edward, D.G. and E.A. Freundt. 1969. Proposal for classifying organisms Leach, R.H. 1973. Further studies on classification of bovine strains of related to Mycoplasma laidlawii in a family Sapromycetaceae, genus Sap- Mycoplasmatales, with proposals for new species, Acholeplasma modicum romyces, within the Mycoplasmatales. J. Gen. Microbiol. 57: 391–395. and Mycoplasma alkalescens. J. Gen. Microbiol. 75: 135–153. Edward, D.G. and E.A. Freundt. 1970. Amended nomenclature for Leach, R.H. 1983. Preservation of Mycoplasma cultures and culture col- strains related to Mycoplasma laidlawii. J. Gen. Microbiol. 62: 1–2. lections. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Fabricant, J. and E.A. Freundt. 1967. Importance of extension and Tully). Academic Press, New York, pp. 197–204. stand­ardization of laboratory tests for the identification and classifi- Lee, G.Y. and G.E. Kenny. 1984. Immunological heterogeneity of super- cation of mycoplasma. Ann. N. Y. Acad. Sci. 143: 50–58. oxide dismutases in the Acholeplasmataceae. Int. J. Syst. Bacteriol. 34: Gardella, R.S., R.A. Del Giudice and J.G. Tully. 1983. Immunofluores- 74–76. cence. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Lewis, J. and J. Poland. 1978. Sensitivity of mycoplasmas of the respira- Tully). Academic Press, New York, pp. 431–439. tory tract of pigs and horses to erythromycin and its use in selective Garwes, D., B. Pike, S. Wyld, D. Pocock and R. Gourlay. 1975. Character- media. Res. Vet. Sci. 24: 121–123. ization of Mycoplasmatales virus-laidlawii 3. J. Gen. Virol. 29: 11–24. Liska, B. 1972. Isolation of a new Mycoplasmatales virus. Stud. Biophys. Gourlay, R.N. 1970. Isolation of a virus infecting a strain of Mycoplasma 34: 151–155. laidlawii. Nature 225: 1165. Lynch, R.E. and B.C. Cole. 1980. Mycoplasma pneumoniae: a prokary- Gourlay, R.N. 1971. Mycoplasmatales virus-laidlawii 2, a new virus isolated ote which consumes oxygen and generates superoxide but which from Acholeplasma laidlawii. J. Gen. Virol. 12: 65–67. lacks superoxide dismutase. Biochem. Biophys. Res. Commun. 96: Gourlay, R.N. 1972. Isolation and characterization of mycoplasma 98–105. viruses. Proceedings of the CIBA Found. Symp., pp. 145–156. Maniloff, J. 1992. Mycoplasma viruses. In Mycoplasmas: Molecular Biol- Gourlay, R.N. 1973. Mycoplasma viruses: isolation, physicohemical, and ogy and Pathogenesis (edited by Maniloff, McElhaney, Finch and biological properties. Ann. N. Y. Acad. Sci. 225: 144–148. Baseman). American Society for Microbiology, Washington, DC, Gourlay, R.N. 1974. Mycoplasma viruses: isolation, physicochemical, and pp. 41–59. biological properties. CRC Crit. Rev. Microbiol. 3: 315–331. Maniloff, J., J. Das and J.R. Christensen. 1977. Viruses of mycoplasmas Gourlay, R.N., J. Brownlie and C.J. Howard. 1973. Isolation of and spiroplasmas. Adv. Virus Res. 21: 343–380. T-­mycoplasmas from goats, and the production of subclinical mastitis Mayberry, W.R., P.F. Smith and T.A. Langworthy. 1974. Heptose-contain- in goats by the intramammary inoculation of human T-mycoplasmas. ing pentaglycosyl diglyceride among the lipids of Acholeplasma modi- J. Gen. Microbiol. 76: 251–254. cum. J. Bacteriol. 118: 898–904. Haberer, K., G. Klotz, J. Maniloff and A. Kleinschmidt. 1979. Structural McCoy, R.E., H.G. Basham, J.G. Tully, D.L. Rose, P. Carle and J.M. Bové. and biological properties of mycoplasmavirus MVL3: an unusual 1984. Acholeplasma florum, a new species isolated from plants. Int. J. virus-procaryote interaction. J. Virol. 32: 268–275. Syst. Bacteriol. 34: 11–15. Heyward, J.T., M.Z. Sabry and W.R. Dowdle. 1969. Characterization of Nakagawa, T., T. Uemori, K. Asada, I. Kato and R. Harasawa. 1992. Mycoplasma species of feline origin. Am. J. Vet. Res. 30: 615–622. Acholeplasma laidlawii has tRNA genes in the 16S–23S spacer of the Hill, A. 1992. Acholeplasma cavigenitalium sp. nov., isolated from the rRNA operon. J. Bacteriol. 174: 8163–8165. vagina of guinea pigs. Int. J. Syst. Bacteriol. 42: 589–592. Neimark, H.C., J.G. Tully, D. Rose and C. Lange. 1992. Chromosome size Hill, A., A. Polak-Vogelzang and A. Angulo. 1992. Acholeplasma multilo- polymorphism among mollicutes. Int. Org. Mycoplasmol. Lett. 2: 261. cale sp. nov., isolated from a horse and a rabbit. Int. J. Syst. Bacteriol. O’Brien, S.J., J.M. Simonson, M.W. Grabowski and M.F. Barile. 1981. 42: 513–517. Analysis of multiple isoenzyme expression among twenty-two species Ichimaru, H. and M. Nakamura. 1983. Biological properties of a plaque- of Mycoplasma and Acholeplasma. J. Bacteriol. 146: 222–232. inducing agent obtained from Acholeplasma oculi. Yale J. Biol. Med. Ogata, M., H. Atobe, H. Kushida and K. Yamamoto. 1971. In vitro sen- 56: 761–763. sitivity of mycoplasmas isolated from various animals and sewage to Johansson, K-E. 1974. Fractionation of membrane proteins from Achole- antibiotics and nitrofurans. J. Antibiot. (Tokyo) 24: 443–451. plasma laidlawii by preparative agarose suspension electrophoresis. Pollack, J.D., J. Banzon, K. Donelson, J.G. Tully, Jr, J.W. Davis, K.J. In Protides of the Biological Fluids – 21st Colloquium (edited by ­Hackett, C. Agbanyim and R.J. Miles. 1996a. Reduction of benzyl Peeters). Pergamon Press, Oxford, pp. 151–156. viologen distinguishes genera of the class Mollicutes. Int. J. Syst. Bac- Johansson, K.E., Pettersson B. 2002. Taxonomy of Mollicutes. In Molecu- teriol. 46: 881–884. lar Biology and Pathogenicity of Mycoplasmas (edited by Razin and Pollack, J.D., M.V. Williams, J. Banzon, M.A. Jones, L. Harvey and Herrmann). Kluwer Academic/Plenum Press, New York, pp. 1–30. J.G. Tully. 1996b. Comparative metabolism of Mesoplasma, Ento- Kato, H., T. Murakami, S. Takase and K. Ono. 1972. Sensitivities in vitro moplasma, Mycoplasma, and Acholeplasma. Int. J. Syst. Bacteriol. 46: to antibiotics of Mycoplasma isolated from canine sources. Jpn. J. Vet. 885–890. Sci. 34: 197–206. Razin, S., J. Tully, D. Rose and M. Barile. 1983. DNA cleavage patterns as Kirby, T., J. Blum, I. Kahane and I. Fridovich. 1980. Distinguishing indicators of genotypic heterogeneity among strains of Acholeplasma between manganese-containing and iron-containing superoxide and Mycoplasma species. J. Gen. Microbiol. 129: 1935–1944. dismutases in crude extracts of cells. Arch. Biochem. Biophys. 20: Rose, D.L. and J.G. Tully. 1983. Detection of b-d-glucosidase: hydrolysis 551–555. of esculin and arbutin. In Methods in Mycoplasmology (edited by Kirchhoff, H. 1978. Acholeplasma equifetale and Acholeplasma hippikon, Tully). Academic Press, New York, pp. 385–389. two new species from aborted horse fetuses. Int. J. Syst. Bacteriol. Rose, D.L., J.G. Tully and R.A. Del Giudice. 1980. Acholeplasma morum, 28: 76–81. a new non-sterol-requiring species. Int. J. Syst. Bacteriol. 30: 647– Kisary, J. and L. Stipkovits. 1975. Effect of Mycoplasma gallinarum on the 654. replication in vitro of goose parvovirus strain “B”. Acta Microbiol. Rottem, S. and O. Markowitz. 1979. Unusual positional distribution of Acad. Sci. Hung. 22: 305–307. fatty acids in phosphatidylglycerol of sterol-requiring mycoplasmas. Kisary, J., A. El-Ebeedy and L. Stipkovits. 1976. Mycoplasma infection of FEBS Lett. 107: 379–382. geese. II. Studies on pathogenicity of mycoplasmas in goslings and Sabin, A.B. 1941. The filterable microorganisms of the pleuropneumo- goose and chicken embryos. Avian Pathol. 5: 15–20. nia group. Bacteriol. Rev. 5: 1–66. 696 Family II. Incertae sedis

Smith, P. and T. Langworthy. 1979. Existence of carotenoids in Achole- Tully, J.G. 1996. Mollicute-host interrelationships: current concepts and plasma axanthum. J. Bacteriol. 137: 185–188. diagnostic implications. In Molecular and Diagnostic Procedures in Somerson, N., J. Kocka, D. Rose and R. Del Giudice. 1982. Isolation Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, of acholeplasmas and a mycoplasma from vegetables. Appl. Environ. San Diego, pp. 1–21. Microbiol. 43: 412–417. Tully, J. and S. Razin. 1970. Acholeplasma axanthum, sp. n.: a new ste- Stephens, E.B., G.S. Aulakh, D.L. Rose, J.G. Tully and M.F. Barile. rol-nonrequiring member of the Mycoplasmatales. J. Bacteriol. 103: 1983a. Intraspecies genetic relatedness among strains of Acholeplasma 751–754. laidlawii and of Acholeplasma axanthum by nucleic acid hybridization. Tully, J.G., D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett and R.F. Whit- J. Gen. Microbiol. 129: 1929–1934. comb. 1988. Acholeplasma entomophilum sp. nov. from gut contents of Stephens, E.B., G.S. Aulakh, D.L. Rose, J.G. Tully and M.F. Barile. 1983b. a wide-range of host insects. Int. J. Syst. Bacteriol. 38: 164–167. Interspecies and intraspecies DNA homology among established spe- Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised tax- cies of Acholeplasma: a review. Yale J. Biol. Med. 56: 729–735. onomy of the class Mollicutes – proposed elevation of a monophyletic Switzer, W.P. 1964. Mycoplasmosis. In Diseases of Swine, 2nd edn (edited cluster of arthropod-associated mollicutes to ordinal rank (Ento- by Dunne). Iowa State University Press, Ames, IA, pp. 498–507. moplasmatales ord. nov.), with provision for familial rank to separate Tanaka, R., A. Muto and S. Osawa. 1989. Nucleotide sequence of species with nonhelical morphology (Entomoplasmataceae fam. nov.) ­tryptophan tRNA gene in Acholeplasma laidlawii. Nucleic Acids Res. 17: from helical species (Spiroplasmataceae), and emended descriptions 5842. of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Tanaka, R., Y. Andachi and A. Muto. 1991. Evolution of tRNAs and tRNA ­Bacteriol. 43: 378–385. genes in Acholeplasma laidlawii. Nucleic Acids Res. 19: 6787–6792. Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, Taylor-Robinson, D. 1983. Metabolism inhibition tests. In Methods in J.M. Bové, P. Carle, D.L. Williamson and T.B. Clark. 1994a. Taxonomic Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, descriptions of eight new non-sterol-requiring Mollicutes assigned to New York, pp. 411–421. the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693. Thorns, C. and E. Boughton. 1978. Studies on film production and Tully, J.G., R.F. Whitcomb, D.L. Rose, J.M. Bové, P. Carle, N.L. Som- its specific inhibition, with special reference to Mycoplasma bovis erson, D.L. Williamson and S. Eden-Green. 1994b. Acholeplasma (M. agalactiae var. bovis). Zentralbl. Veterinarmed. B 25: 657–667. brassicae sp. nov. and Acholeplasma palmae sp. nov., two non-sterol- Tully, J.G. 1973. Biological and serological characteristics of the achole- requiring mollicutes from plant surfaces. Int. J. Syst. Bacteriol. 44: plasmas. N. Y. Acad. Sci. 225: 74–93. 680–684. Tully, J.G. 1979. Special features of the acholeplasmas. In The Mycoplas- Waites, K.B., J.G. Tully, D.L. Rose, P.A. Marriott, R.O. Davis and mas, vol. 1 (edited by Barile and Razin). Academic Press, New York, G.H. Cassell. 1987. Isolation of Acholeplasma oculi from human amni- pp. 431–449. otic fluid in early pregnancy. Curr. Microbiol. 15: 325–327. Tully, J.G. 1983. Methods in mycoplasmology, vol. 2, Diagnostic Myco- Whitcomb, R.F. and D.L. Williamson. 1975. Helical wall-free prokary- plasmology. Academic Press, New York. otes in insects: multiplication and pathogenicity. Ann. N. Y. Acad. Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sor- Sci. 266: 260–275. bitan growth requirements of mollicutes. In Molecular and Diagnos- Whitcomb, R.F., J.G. Tully, J.M. Bové and P. Saglio. 1973. Spiroplas- tic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). mas and acholeplasmas: multiplication in insects. Science 182: Academic Press, San Diego, pp. 381–389. 1251–1253.

Family II. Incertae sedis This family includes the phytoplasma strains of the order these plant pathogens and symbionts have been well studied by Acholeplasmatales. Although never ­cultured in cell-free media, culture-­independent methods.

Genus I. “Candidatus Phytoplasma” gen. nov. IRPCM Phytoplasma/Spiroplasma Working Team 2004, 1244

Ni g e l A. Ha rr i s o n , Da w n Gu n d e r s e n -Ri n d a l a n d Ro b e r t E. Da v i s Phy.to.plas¢ma. Gr. masc. n. phytos a plant; Gr. neut. n. plasma something formed or molded, a form. Phytoplasmas (Sears and Kirkpatrick, 1994) are wall-less, nutri- media has not yet been demonstrated for any phytoplasma. tionally fastidious, phytopathogenic prokaryotes 0.2–0.8 µm Their genome sizes have been estimated to range from 530 to in diameter that morphologically resemble members of the 1350 kb, and the G+C content of phytoplasma DNA is about Mollicutes. Sequencing of nearly full-length PCR-amplified 23–30 mol%. The presence of a characteristic oligonucleotide 16S rRNA genes (Gundersen et al., 1994; Namba et al., 1993; sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG Seemüller et al., 1994), combined with earlier studies (Kuske CYG GDC T, and standard codon usage indicate that phytoplas- and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the mas represent a distinct taxon for which the name “Candidatus first comprehensive phylogeny of the organisms and showed Phytoplasma” has been adopted by specialists in the molecular that they constitute a unique, monophyletic clade within the biology and pathogenicity of these and similar phytopatho- Mollicutes. These organisms are most closely related to mem- genic organisms (IRPCM Phytoplasma/Spiroplasma Working bers of the genus Acholeplasma within the Anaeroplasma clade as Team – Phytoplasma Taxonomy Group, 2004). At present, the defined by Weisburg et al. (1989). Sustained culture in cell-free designation “Candidatus” must still be used for new types. Genus I. “Candidatus Phytoplasma” 697

Further descriptive information for the uptake of water and nutrients (Flores et al., 1999). Phytoplasma cells typically have a diameter less than 1 µm Phloem sap is unique in that it contains from 12 to 30% sucrose and are polymorphic. Viewed in ultra-thin section by electron and is under high hydrostatic (turgor) pressure (Evert, 1977). microscopy Figure 115, they appear ovoid, oblong, or filamen- Sieve elements have pores in their end plates and lateral walls, tous in plant and insect hosts (Doi et al., 1967; Hearon et al., allowing passage of photosynthate to adjacent sieve tube ele- 1976). Transmission electron microscopy of semi-thick (0.3 µm) ments. The sieve pores, which have an average diameter of ~0.2 sections (Thomas, 1979) and serial sections (Chen and Hiruki, µm, are of sufficient size to allow passage of spherical and fila- 1978; Florance and Cameron, 1978; Waters and P. Hunt., 1980), mentous phytoplasma cells from one sieve element to another and scanning electron microscopy studies (Bertaccini et al., (McCoy, 1979). The chemical composition of sieve sap is com- 1999; Haggis and Sinha, 1978; Marcone et al., 1996) have done plex, containing sugars, minerals, free amino acids, proteins, much to clarify the gross cellular morphology of phytoplasmas. and ATP (Van Helden et al., 1994). This rich milieu, with its They range from spherical to filamentous, often with exten- high osmotic and hydrostatic pressures, serves to support exten- sive branching reminiscent of that seen in Mycoplasma mycoides. sive multiplication of phytoplasmas in planta. Phytoplasmas also Small dense rounded forms ~0.1 µm in diameter, formerly con- multiply in the internal tissues and organs of their insect vectors sidered to be “elementary bodies” when seen in thin section, (Kirkpatrick et al., 1987; Lefol et al., 1994; Marzachi et al., 2004; were shown to represent constrictions in filamentous forms. Nasu et al., 1970), which are primarily leafhoppers, planthop- Dumbbell-shaped forms once thought to be “dividing cells” pers, and psyllids (D’Arcy and Nault, 1982; Jones, 2002; Wein- are actually branch points of filamentous forms, whereas forms traub and Beanland, 2006). In many respects, the composition thought to have internal vesicles have been shown to have invo- of insect hemolymph is similar to that of plant phloem sap, as luted membranes oriented such that the plane of the sections both contain high levels of complex and simple organic com- cut through the cell membrane twice (McCoy, 1979). Phyto- pounds (Moriwaki et al., 2003; Saglio and Whitcomb, 1979). plasma cell membranes are resistant to digitonin and sensitive Physical maps of several phytoplasma genomes have been to hypotonic salt solutions, and, as such, are similar to those of constructed (Firrao et al., 1996; Lauer and Seemüller, 2000; non-sterol requiring mollicutes (Lim et al., 1992). Marcone and Seemüller, 2001; Padovan et al., 2000). The pres- Phytoplasmas are consistently observed within phloem sieve ence of extrachromosomal DNAs or plasmids in numerous elements (Christensen et al., 2004; McCoy et al., 1989; Oshima phytoplasmas has also been reported (Davis et al., 1988; Denes et al., 2001b; Webb et al., 1999) and occasionally have been and Sinha, 1991; Kuboyama et al., 1998; Kuske and Kirkpat- reported in both companion cells (Rudzinska-Langwald and rick, 1990; Liefting et al., 2004; Lin et al., 2009; Nakashima and Kaminska., 1999; Sears and Klomparens, 1989) and paren- Hayashi, 1997; Nishigawa et al., 2003; Oshima et al., 2001a; Tran- chyma cells (Esau et al., 1976; Siller et al., 1987) of infected Nguyen and Gibb, 2006) and suggested as a potential means of plants. Sieve elements are specialized living cells that lack nuclei intermolecular recombination (Nishigawa et al., 2002b). Phyto- when mature and transport photosynthate from leaves not only plasma-associated extrachromosomal DNAs have been shown to growing tissues, but also to other tissues unable to photo- to contain genes encoding a putative geminivirus-related rep- synthesize (Oparka and Turgeon, 1999; Sjölund, 1997). This lication (Rep) protein (Liefting et al., 2006; Nishigawa et al., applies particularly to roots that require considerable energy 2001; Rekab et al., 1999) and a single-stranded DNA-binding

FIGURE 115. Electron micrographs of ultrathin sections of leaf petiole from a sunnhemp (Crotalaria juncea L.) plant displaying Crotalaria phyllody disease symptoms. (a) Polymorphic phytoplasma cells occluding the lumen of adjacent leaf phloem sieve tube elements. Bar = 2 µm. (b) Ultratructural morphology indicates phytoplasma cells are bounded by a unit membrane and contain DNA fibrils and ribosomes. Bar = 200 nm. Images provided by Phil Jones. 698 Family II. Incertae sedis protein (Nishigawa et al., 2002a), as well as a putative gene simi- The ­chromosome of “Candidatus­ Phytoplasma mali” is charac- lar to DNA primase of other bacterial chromosomes (Liefting terized by large terminal inverted repeats and covalently closed et al., 2004) and still other genes of as yet unknown identity. hairpin ends. Analysis of protein-coding genes revealed that gly- Moreover, heterogeneity in extrachromosomal DNAs has been colysis, the major energy-yielding pathway supposed for OY-M associated with reduced pathogenicity and loss of insect vector phytoplasma, is incomplete in AT phytoplasma. It also differs transmissibility (Denes and Sinha, 1992; Nishigawa et al., 2002a, from OY-M and AY-WB phytoplasmas by a lower G+C content 2003). (21.4 mol%), fewer paralogous genes, a strongly reduced num- Onion yellows mild strain (OY-M) was the first phytoplasma ber of ABC transporters for amino acids, and an extended set genome to be completely sequenced. The genome of this of genes for homologous recombination, excision repair, and aster yellows group strain consists of a circular chromosome SOS response. of 860,632 bp. It also contains two extrachromosomal DNAs, Comparative genomics have also recently identified ORFs EcOYM (5025 bp) and plasmid pOYM (3932 bp) (Nishigawa shared by AY-WB phytoplasma and the distantly-related corn et al., 2003; Oshima et al., 2002), representing two different stunt pathogen Spiroplasma kunkelii that are absent from obligate classes based on the type of replication protein encoded. While animal and human pathogenic mollicutes. These proteins were EcOYM contains a rep gene homologous to that of the gemi- identified as polynucleotide phosphorylase (PNPase), cmp- niviruses, pOYM has a rep gene that encodes a unique protein binding factor (CBF), cytosine deaminase, and Y1xR protein with characteristics of both viral-rep and plasmid-rep (Namba, and could be important for insect transmission or plant patho- 2002). The chromosome is a circular DNA molecule with a G+C genicity. Also identified were four additional proteins, ppGpp content of 28 mol% and contains 754 open reading frames synthetase, HAD hydrolase, AtA (AAA type ATPase), and P-type (ORFs), comprising 73% of the chromosome. Of these, 66% of Mg2+ transport ATPase, that seemed to be more closely related ORFs exhibit significant homology to gene sequences currently between AY-WB and Spiroplasma kunkelii than to their mycoplas- archived in the GenBank database. Putative proteins encoded mal counterparts (Bai et al., 2004). by ORFs could be assigned to one of six different functional Phytoplasmas possess a unique genome architecture that is categories: (1) information storage and processing (260 ORFs); characterized by multiple, nonrandomly distributed sequence- (2) metabolism (107 ORFs); (3) cellular processes (77 ORFs); variable mosaics (SVMs) of clustered genes, originally rec- (4) poorly characterized, i.e., with homology to uncharacter- ognized in a study of closely related “Candidatus Phytoplasma ized proteins of other organisms (50 ORFs); or (5) others, i.e., asteris”-related strains CPh and OY-M (Jomantiene and Davis, without homology to any known proteins (260 ORFs). Like 2006). Targeted genome sequencing and comparative genomics mycoplasmal genomes, the OY-M phytoplasma genome lacks indicated that this genome architecture is a common charac- many genes related to amino acid and fatty acid biosynthesis, teristic among phytoplasmas, leading to the proposal that the the tricarboxylic acid cycle, and oxidative phosphorylation. origin of SVMs was an ancient event in the evolution of the phy- However, OY-M phytoplasma differs from mycoplasma in that it toplasma clade (Jomantiene et al., 2007), perhaps as a result of lacks genes for the phosphotransferase system and for metabo- recurrent targeted attacks by mobile elements such as phages lizing UDP-galactose to glucose 1-phosphate, suggesting that (Wei et al., 2008a). Jomantiene and Davis (2006) proposed that it possesses a unique sugar intake and metabolic system. Fur- sizes and numbers of SVMs could account in part for the known thermore, OY-M phytoplasma lacks most of the genes needed variation in genome size among phytoplasma strains; this con- to synthesize nucleotides and ATP suggesting that it probably cept was independently suggested by Bai et al. (2006) on the assimilates these and other necessary metabolites from host basis of results from a comparative study of two completely cytoplasm. Many genes, such as those for glycolysis, are pres- sequenced phytoplasma genomes. Nucleotide sequences within ent as multiple redundant copies representing 18% of the total SVMs included full length or pseudogene forms of fliA, an ATP- genome. Twenty-seven genes encoding transporter systems ­dependent Zn protease gene, tra5, smc, himA, tmk, and ssb (encod- such as malate, metal-ion and amino acid transporters, some ing single-stranded DNA-binding protein), genes potentially of which have multiple copies, were identified, suggesting that encoding hypothetical proteins of unknown function, genes phytoplasmas aggressively import many metabolites from the exhibiting similarities to transposase, and a phage-related gene host cell. Other than genes encoding glucanase and hemolysin- (Jomantiene­ et al., 2007). A similar set of nucleotide sequences like proteins, no other genes presently known to be related to occurs in AY-WB genomic regions termed potential mobile units bacterial pathogenicity were evident in the OY-M phytoplasma (PMUs) by Bai et al. (2006). The presence of sequences encod- genome, suggesting novel mechanisms for virulence. ing putatively secreted and/or transmembrane, cell surface- Annotation of the OY-M phytoplasma genome has been interacting proteins indicates that these genomic features are followed by three other phytoplasma genome annotations. likely to be significant for phytoplasma/host interactions (Bai Aster yellows witches’-broom phytoplasma (“Candidatus Phy- et al., 2006; Jomantiene and Davis, 2006; Jomantiene et al., toplasma asteris”-related strain AY-WB) possesses a circu- 2007). lar 706,569 nucleotide chromosome and plasmids AYWB-pI Short (17–35 bases) conserved, imperfect palindromic DNA (3872 bp), -pII (4009 bp), -pIII (5104 bp), and -pIV (4316 sequences (PhREPs) that are present in SVMs possibly play a bp) (Bai et al., 2006). Australian tomato big bud phytoplasma role in phytoplasma genome plasticity and targeting of mobile (“Candidatus­ Phytoplasma australiense”-related strain TBB) genetic elements. SVMs can be viewed as composites formed has a circular 879,324 bp chromosome and a 3700 bp plas- by the acquisition of genes through horizontal transfer, recom- mid (Tran-Nguyen et al., 2008), whereas apple proliferation bination, and rearrangement, and capture of mobile elements phytoplasma (“Candidatus Phytoplasma mali”-related strain recurrently targeted to SVMs, leading Jomantiene et al. (2007) AT) has a linear 601,943 bp chromosome (Kube et al., 2008). to suggest that SVMs provide loci for acquisition of new genes Genus I. “Candidatus Phytoplasma” 699 and targeting of mobile genetic elements to specific regions in enzyme-linked immunosorbent (ELISA), immunofluorescence, phytoplasma chromosomes. or Western blot assays. Antigenic similarity revealed among phy- The chromosomes of avirulent, mildly, moderately, and toplasmas by these assays is often in agreement with ­relationships highly virulent strains of “Candidatus Phytoplasma mali” demonstrated by vector transmission ­studies. Detection of anti- (­Seemüller and Schneider, 2007) differ from one another in genically distinct phytoplasmas in plants exhibiting very similar size and exhibit distinct restriction endonuclease patterns when disease symptoms attests to the unreliability of symptom expres- cleaved with rare cutting enzymes. PCR-based DNA ampli- sion alone as a means of differentiating phytoplasmas. fications, primed separately by eight primer pairs, revealed Improvements in phytoplasma extraction methods have target sequence heterogeneity among all “Candidatus Phyto- provided a source of immunogens for monoclonal antibody plasma mali”-related strains tested, but no correlations linked (MAb) production (Chang et al., 1995; Hsu et al., 1990; Jiang ­molecular markers with strain virulence or the maximum titer et al., 1989; Loi et al., 2002, 1998; Shen and Lin, 1993; Tanne obtained upon infection of apple trees. In a separate study, a et al., 2001). Used in ELISA, dot or tissue blot immunoassay, comparison of mild (OY-M) and severe (OY-W) strains of onion ­immunocapture PCR (Rajan et al., 1995), immunofluorescence yellows (OY) phytoplasma indicated that severe symptoms were microscopy, or immunosorbent electron microscopy (ISEM) associated with higher populations of OY-W in infected host (Clark, 1992; Shen and Lin, 1994), MAbs have demonstrated plants (Oshima et al., 2001b). A cluster of eight genes, consid- considerable promise for detection and differentiation of phy- ered essential for glycolysis, were subsequently identified within toplasmas infecting a broad range of host plants, including a similar 30 kb genomic region of both strains (Oshima et al., woody perennials (Guo et al., 1998). Due to their high degree 2007). Of these, five genes (smtA, greA, osmC, eno, and pfkA) were of specificity, monoclonals seem most suited for differentiating randomly duplicated in OY-W, possibly influencing glycolytic very closely related strains (Lee et al., 1993a). activitiy. A higher consumption of metabolites such as sugars in Isolation, cloning, and expression of immunodominant pro- the intracellular environment of the phloem may explain dif- tein genes have identified putative proteins that account for a ferences between OY-W and OY-M in growth rate, which in turn major portion of the membrane proteins of several phytoplas- may be linked, directly or indirectly, to symptom severity. mas (Arashida et al., 2008; Barbara et al., 2002; Berg et al., 1999; Cloned fragments of phytoplasma DNA have been widely Blomquist et al., 2001; Galetto et al., 2008; Kakizawa et al., 2004, employed as probes in dot and Southern blot hybridization 2009; Morton et al., 2003; Suzuki et al., 2006; Yu et al., 1998). assays to identify and characterize phytoplasmas (reviewed by When these purified proteins were used as immunogens, the Lee and Davis, 1992; Lee et al. (2000). Southern blot restriction resulting polyclonal antisera exhibited high specific titers and low fragment length polymorphism (RFLP) analysis has enabled background reactions in ELISA and Western blot analyses that investigations of genetic relationships among phytoplasmas were designed to detect phytoplasma proteins in infected hosts. associated with similar hosts or with symptomatologically similar Similarly, the secA gene was cloned from an onion yellows (OY-M) diseases (Kison et al., 1997, 1994; Kuske et al., 1991; Schneider strain of aster yellows phytoplasma (Kakizawa et al., 2001) and and Seemüller, 1994b). Several discrete phytoplasma groups, used to raise an anti-SecA rabbit antibody against a purified par- each comprising strains that shared extensive sequence homol- tial SecA protein expressed in Escherichia coli. Light microscopy ogy and little or no apparent homology with other phytoplas- of thin sections of garland chrysanthemum (Chrysanthemum coro- mas, were identified by this type of analysis. Lee and co-workers narium) treated by immunohistochemical straining revealed that (1992) coined the term “genomic strain cluster” to denote the SecA protein was present in phloem of OY-M-infected but not each of seven discrete genotypic groups resolved by employing healthy host plants. In addition, antisera against both OY-M phy- a selection of phytoplasma genomic DNA probes (reviewed by toplasma SecA protein and GyrA protein of Acholeplasma laidlawii Lee and Davis, 1992). Of these, aster yellows (AY) was the largest reacted with proteins of several unrelated phytoplasmas extracted group, represented by 15 genetically variable strains that were from plant tissues (Koui et al., 2002; Wei et al., 2004a). further delineated into three distinct genomic types (types I, II, Phytoplasmas are the apparent etiological agents of diseases and III) or subclusters (Lee et al., 1992). Significantly, major of at least 1000 plant species worldwide (McCoy et al., 1989; groupings later revealed by RFLP analysis of 16S rRNA genes Seemüller et al., 1998). Although they can be transmitted from were consistent with those defined by monoclonal antibody infected to healthy plants by scion or root grafts, most plant typing (Lee et al., 1993a) and other molecular methods (Lee to plant spread occurs naturally via phloem-feeding insect vec- et al., 1998b), but differed from distinctions made in traditional tors primarily of the family Cicadellidae (leafhoppers) and, less classification based solely on biological properties such as plant commonly, by planthoppers (Fulgoroidea) of the family Ciixidae host range, symptomatology, and insect vector specificity and psyllids (Psylloidea) (D’Arcy and Nault, 1982; Tsai, 1979; (­Chiykowski and Sinha, 1990). Weintraub and Beanland, 2006). Phytoplasmas are transmit- Polyclonal antibodies (PAbs) have been produced against ted in a circulative-propagative manner that typically involves phytoplasma-enriched extracts (intact organisms or membrane a transmission latent period from 2 to 8 weeks (Carraro et al., fractions) partially purified from plants (reviewed by Chen 2001; Webb et al., 1999). The insect vector becomes infected et al., 1989) and against vector leafhopper-derived immunogens upon ingesting phytoplasmas in phloem sap of infected plants. (Errampelli and Fletcher, 1993; Kirkpatrick et al., 1987). Most After an incubation period of one to several weeks, the phyto- PAbs exhibit relatively high background reactions with healthy plasma multiplies to high titer in the salivary glands and the host antigens; thus, generation of useful polyclonal antisera has insect becomes capable of infecting the phloem of the healthy been limited so far to a few phytoplasmas maintained at high plants on which it feeds (Kunkel, 1926; Lee et al., 1998a; Nasu titer in host tissues. Phytoplasmas can be differentiated on the et al., 1970). Generally, phytoplasma infection does not appear basis of their antigenic properties through the use of PAbs in to significantly affect the activity, weight, longevity, or fecundity 700 Family II. Incertae sedis of vector insects (Garnier et al., 2001). Some phytoplasmas can as periwinkle (Catharanthus roseus) and in certain woody peren- be vectored by many species of leafhoppers (McCoy et al., 1989; nial hosts such as alder (Alnus) and most poplar (Populus) spe- Nielson, 1979) and different insect species may serve as vectors cies. Lowest phytoplasma concentrations, from 370 to 34,000 in different geographic regions. Several vectors also have the cells per gram of tissue, were detected in apple trees that were ability to transmit more than one type of phytoplasma, whereas grafted on resistant rootstocks and in oak (Quercus robur) or other phytoplasmas are transmitted by one or a few vector spe- hornbeam (Carpinus betulus) trees exhibiting nonspecific leaf cies to a narrow range of plant species (Lee et al., 1998a). There yellowing symptoms (Berg and Seemüller, 1999). is mounting evidence also for transovarial transmission of some Colonization is usually marked by phloem dysfunction and phytoplasmas (Alma et al., 1997; Hanboonsong et al., 2002; a reduction in photosynthetic capacity. Alterations in phloem Kawakita et al., 2000; Tedeschi et al., 2006). function have been correlated with structural degeneration of Plants may serve as both natural and experimental hosts to sieve elements due possibly to physical blockage by colonizing several different phytoplasmas. Dual or mixed infections involv- phytoplasma or the action of a phytotoxin (Guthrie et al., 2001; ing related or unrelated phytoplasmas are known to occur Siddique et al., 1998). The onset of symptoms may be accom- naturally in plants and appear to be more common in peren- panied by substantial impairment of the photosynthetic rate of nial than annual plants (Bianco et al., 1993; Lee et al., 1995). mature leaves and by fluctuations in carbohydrate and amino Also, closely related phytoplasma strains are capable of induc- acid levels in source versus sink leaves (Lepka et al., 1999; Tan ing dissimilar symptoms on the same plant species (White et al., and Whitlow, 2001). Leaf yellowing is associated with: decreases 1998), whereas similar symptoms on the same host plant may be in chlorophyll content, carotenoids, and soluble proteins (Ber- induced by unrelated phytoplasmas (Harrison et al., 2003). The tamini and Nedunchezhian, 2001); abnormal stomatal func- ability to accurately identify phytoplasmas by using DNA-based tion (Martinez et al., 2000); histopathological changes such the methods has shown that these organisms are more genetically amount of total polyphenols; loss of cellular integrity (Musetti diverse than was once thought (Davis and Sinclair, 1998). The et al., 2000); fluctuations in hydrogen peroxide; peroxidase geographic occurrence of phytoplasmas is determined largely activity and glutathione content in diseased versus healthy plant by geographic ranges and feeding behavior (mono-, oligo-, or tissues (Musetti et al., 2004); and increases in calcium (Ca2+) polyphagous) of the vector species, the relative susceptibility of ions in cells (Musetti and Favali, 2003; Rudzinska-Langwald the preferred host plant species, and the native host ranges of and Kaminska, 2003). Such adverse changes are accompanied plant and insect hosts (Lee et al., 1998a). Phytoplasmas can be by differential regulation of genes encoding proteins involved introduced into new geographic regions by long-distance dis- in floral development (Pracros et al., 2006), photosynthesis, persal of infectious vectors (Lee et al., 2003) and by movement sugar transport, and response to stress or in pathways of lipid of infected plants or vegetative plant parts. Most recently, phy- and phenylpropanoid or phytosterol synthesis (Albertazzi toplasma DNA has been detected in embryos of aborted seed et al., 2009; Carginale et al., 2004; Hren et al., 2009; Jagoueix- from diseased plants (Cordova et al., 2003; Nipah et al., 2007) ­Eveillard et al., 2001). and seed transmission of phytoplasmas infecting alfalfa (Medi- The organisms degenerate and lose their cellular contents cago sativa L.) has been demonstrated (Khan et al., 2002). following treatment of infected plants with tetracycline antibi- An array of characteristic symptoms is associated with phy- otics (Kamin´ska and S´liwa, 2003; Sinha and Peterson, 1972). toplasma infection of several hundred plant species world- Tetracycline sensitivity and the lack of sensitivity to cell wall- wide. Symptoms vary according to the particular host species, inhibiting antibiotics such as penicillin (Davis and Whitcomb, stage of host infection and the associated phytoplasma strain 1970; Ishii et al., 1967) also support their inclusion in the Mol- (reviewed by Davis and Lee, 1992; Hogenhout et al., 2008; licutes. Protective or therapeutic treatments with tetracycline Kirkpatrick, 1989, 1992; Lee et al., 2000; McCoy et al., 1989; antibiotics for phytoplasma disease control have been extended Seemüller et al., 2002; Sinclair et al., 1994). Some symptoms to a few high-value crop plants such as coconut for control of indicative of profound disturbances in the normal balance of palm lethal yellowing, and to cherries and peaches for control growth ­regulators in plants include virescence (greening of pet- of X-disease (McCoy, 1982; Nyland, 1971; Raju and Nyland, als), phyllody (conversion of floral organs into leafy structures), 1988). Administered by trunk injection, treatment of each tree big bud, floral proliferation, sterility of flowers, proliferation with 1.0 g (protective dose) or 3.0 g (therapeutic dose) three of adventitious or axillary shoots, internode elongation and times per year was sufficient for control of coconut lethal yel- ­etiolation, generalized stunting (small flowers, leaves and fruits lowing disease (McCoy, 1982). or shortened internodes), unseasonal discoloration of leaves or shoots (yellow to purple discoloration), leaf curling, cupping Enrichment and isolation procedures or crinkling, witches’-brooms (bunchy growth at stem apices), Isolation of phytoplasma-enriched fractions from plant and vein clearing, vein enlargement, phloem discoloration, and gen- insect host tissues is possible by differential centrifugation and eral plant decline such as die-back of twigs, branches and trunks filtration after first disrupting tissues in osmotically-augmented (Lee and Davis, 1992; Lee et al., 2000; McCoy et al., 1989). buffers (Kirkpatrick et al., 1995; Lee et al., 1988; Sinha, 1979; Infection of herbaceous host plants is followed by rapid Thomas and Balasundaran, 2001). Further purification of phy- intraphloemic spread of phytoplasma from leaves to roots, often toplasmas is possible by centrifugation of enriched preparations accompanied by six-fold increases in phytoplasma populations in discontinuous Percoll density gradients (Davis et al., 1988; in these tissues between 14 and 28 d post-inoculation (Kuske Gomez et al., 1996; Jiang and Chen, 1987) or by affinity chro- and Kirkpatrick, 1992a; Wei et al., 2004b). Phytoplasma con- matography using phytoplasma-specific antibodies coupled to centrations ranging from 2.2 × 108 to 1.5 × 109 cells per gram of Protein A-Sepharose columns (Jiang et al., 1988; Seddas et al., tissue have been measured in high titer herbaceous hosts such 1995). Viability of these enriched preparations may be assessed Genus I. “Candidatus Phytoplasma” 701 by infectivity tests in which aliquots of the phytoplasma prepa- a gene encoding an RNase P ribozyme (Wagner et al., 2001), rations are micro-injected into vector insects, which are then recA (Chu et al., 2006), rpoC (Lin et al. 2006), polC (Chi and fed on healthy indicator plants (Nasu et al., 1974; Sinha, 1979; Lin., 2005), and insertion sequence (IS)-like elements (Lee Whitcomb et al., 1966a, b). Separation of enriched phytoplasma et al., 2005). Numerous other putative genes or pseudogenes DNA from mixtures with host DNA is also possible by use of have been identified recently after partially or fully sequencing cesium chloride-bisbenzimide buoyant density gradient centrif- random fragments of genomic DNA cloned from phytoplasmas ugation (Kollar and Seemüller, 1989). Present as an uppermost by various methods (Bai et al., 2004; Cimerman et al., 2006, band in final gradients, phytoplasma DNA fractionated by this 2009; Davis et al., 2003b, 2005; Garcia-Chapa et al., 2004; Lieft- means was suitable for endonuclease digestion and cloning for ing and Kirkpatrick, 2003; Melamed et al., 2003; Miyata et al., DNA probe development (Harrison et al., 1992, 1991; Kollar 2003; Streten and Gibb, 2003). and Seemüller, 1990). Development of phytoplasma-specific rRNA gene primers has permitted PCR-mediated amplification of various regions Maintenance procedures of the rRNA operons (Ahrens and Seemüller, 1992; Baric and Viable phytoplasmas have been maintained for at least 6 years Dalla-Via, 2004; Davis and Lee, 1993; Deng and Hiruki, 1991; in intact vector insects frozen at −70°C (Chiykowski, 1983). ­Gundersen and Lee, 1996; Lee et al., 1993b) (Namba et al., Viable X-disease phytoplasmas have been maintained for 2 1993; Smart et al., 1996). RFLP analysis of PCR-amplified rDNA weeks in salivary glands suspended in a tissue culture medium provided a practical solution to the problem of phytoplasma (Nasu et al., 1974). Extracts of phytoplasma-infected insects identification and classification (Lee et al., 2000, 1998b). Pair- prepared in a MgCl2/glycine buffer, osmotically adjusted to wise comparisons of disparate strains were marked by consid- 800 milliosmoles/kg with sucrose, retained their infectivity for erable differences in RFLP patterns, whereas strains that were up to 3 d (Smith et al., 1981). Phytoplasma strains have been considered closely related on the basis of similar biological routinely maintained in diseased plants kept in an insect-proof properties were often, although not always, indistinguishable on greenhouse or in plantlets grown in tissue culture (Bertaccini the basis of RFLP patterns. Alternatively, heteroduplex mobility et al., 1992; Davis and Lee, 1992; Jarausch et al., 1996; Sears analysis has demonstrated greater sensitivity than RFLP analy- and Klomparens, 1989; Wongkaew and Fletcher, 2004). While sis for detecting minor variability in 16S rRNA genes of closely plant to plant transmission is accomplished naturally by vector related phytoplasma strains (Cousin et al., 1998; Wang and insects and, in some cases, through grafts, experimental trans- Hiruki, 2000), since RFLP analysis is limited to detection of rec- missions commonly include the use of plant parasitic dodders ognition sites for restriction endonucleases. Cluster analysis of (Cuscuta sp.) (Marcone et al., 1999a). Although phytoplasma rDNA RFLP patterns provided the first means to differentiate strains are commonly maintained in plants by periodic graft between known and unknown phytoplasmas from a wide range inoculation, maintenance of phytoplasmas exclusively in plants of plant hosts and geographic locations, and to resolve phyto- can result in strain attenuation over time and an associated loss plasmas into well-defined phylogenetic groups and ­subgroups of transmissibility by vector insects (Chiykowski, 1988; Denes (Ahrens and Seemüller, 1992; Lee et al., 1993b; Schneider and Sinha, 1992). et al., 1993, 1995).

Differentiation of the genus “Candidatus Phytoplasma” Taxonomic comments from other genera The inability to cultivate phytoplasmas outside of their plant Phytoplasma-specific nucleic acid probes and PCR technol- and insect hosts has thus far rendered traditional methods ogy have largely supplanted traditional methods of electron impractical as aids for taxonomy of these organisms. Unlike microscopy and biological criteria for sensitive detection, iden- their culturable Mollicute relatives, which were originally clas- tification, and genetic characterization of phytoplasmas. Molec- sified based only upon biological and phenotypic properties ular-based analyses have shown phytoplasma genomes to be in pure culture, phytoplasmas cannot be classified by these A+T rich (Kollar and Seemuller, 1989; Oshima et al., 2004) and criteria. Through application of DNA-based methods, it is now to range from 530 to 1350 kbp in size (Marcone et al., 1999b, possible to accurately identify and characterize phytoplasmas 2001; Neimark and Kirkpatrick, 1993). Before any phytoplasma and to assess their genetic interrelationships. These capabilities genomes were sequenced, phytoplasmas were shown to con- have assisted development of classification systems, first based tain two rRNA operons (Davis, 2003a; Harrison et al., 2002; Ho on hybridization data, later based on 16S rDNA RFLPs, and ulti- et al., 2001; Jomantiene et al., 2002; Jung et al., 2003a; Lee et al., mately on phylogenetic analysis of 16S rRNA genes and other 1998b; Liefting et al., 1996; Marcone et al., 2000; Schneider conserved gene sequences. Classification schemes founded and Seemüller, 1994a). Other genes that have been identified upon these molecular criteria have been refined and expanded include ribosomal protein genes (Gundersen et al., 1994; Lee upon over time, with the goal of defining a taxonomy for these et al., 1998b; Lim and Sears, 1992; Martini et al., 2007; Miyata unique organisms. In a phytoplasma classification scheme pro- et al., 2002a; Toth et al., 1994) of the S10-spc operon (Miyata posed by Lee et al. (1993b), based on analyses of rDNA RFLPs, et al., 2002a), a nitroreductase gene (Jarausch et al., 1994), a total of nine primary 16S rDNA groups (termed 16Sr groups) DNA gyrase genes (Chuang and Lin, 2000), genes encoding and 14 subgroups were initially recognized. Phytoplasma groups elongation factors G and Tu (An et al., 2006; Berg and Seemül- delineated by these analyses were consistent with genomic ler, 1999; Koui et al., 2003; Marcone et al., 2000; Miyata et al., strain clusters previously identified by DNA hybridization analy- 2002b; Schneider et al., 1997), secA, secY, and secE genes of a sis (Lee et al., 1992), although a greater diversity among strains functional Sec protein translocation system (Kakizawa et al., comprising group 16SrI (aster yellows and related strains) was 2001, 2004), gidA, potB, potC, and potD (Mounsey et al., 2006), indicated by the earlier hybridization data. Subgroups within a 702 Family II. Incertae sedis given 16Sr group were distinguished by the presence of one or gene sequence similarities that were 1.2–2.3% or greater and, more restriction sites in a phytoplasma strain that differed from in some instances, by additional considerations such as plant those in all existing members of a given subgroup. For those host and vector specificity, primer specificity, and RFLP com- strains in which intra-rRNA operon heterogeneity was detected, parisons of ribosomal and nonribosomal DNA, as well as sero- subgroup designations were assigned according to the com- logical comparisons (Seemüller et al., 2002, 1998). bined patterns of both 16S rRNA genes. Most recently, Wei et al. (2007) applied computer-simulated RFLP analysis of more variable ribosomal protein genes RFLP analysis for classification of phytoplasma strains. Through (Gundersen et al., 1994; Lee et al., 2004a, b) or tuf genes comparisons of virtual RFLP patterns of 16S rRNA genes and (Marcone et al., 2000; Schneider et al., 1997) has provided a calculations of coefficients of RFLP similarity, the authors means for more detailed subdivision of phytoplasma primary classified all available 16S rRNA gene sequences, includ- groups delineated by 16S rDNA RFLP data. This strategy for ing sequences from 250 previously unclassified phytoplasma finer subgroup differentiation has been used to modify and strains, into a total of 28 16Sr RFLP groups. These included ten expand upon earlier classifications and to incorporate many new groups and dozens of new subgroup lineages (Cai et al., newly identified phytoplasma strains. Based on RFLP analy- 2008; Wei et al., 2008b). Each new group represents a potential sis of nearly full-length 16S rRNAs, at least 15 primary 16Sr “Candidatus Phytoplasma” species level taxon. This informa- groups have been recognized (Lee et al., 1998b; Montano et al., tion was used to augment the 16Sr RFLP classification system 2001): 16SrI, Aster yellows; 16SrII, Peanut witches’-broom; (Lee et al., 2000, 1998b, 1993b) with the following additional 16SrIII, X-disease; 16SrIV, Coconut lethal yellows; 16SrV, Elm groups: 16SrXVI, Sugarcane yellow leaf syndrome; 16SrXVII, yellows; 16SrVI, Clover proliferation; 16SrVII, Ash yellows; Papaya bunchy top group; 16SrXVIII, American potato purple 16SrVIII, Loofah witches’-broom; 16SrIX, Pigeonpea witches’- top wilt group; 16SrXIX, Japanese chestnut witches’-broom broom; 16SrX, Apple proliferation; 16SrXI, Rice yellow dwarf; group; 16SrXX, Buckthorn witches’-broom group; 16SrXXI, 16SrXII, Stolbur; 16SrXIII, Mexican periwinkle virescence; Pine shoot proliferation group; 16SrXXI, Nigerian coconut 16SrXIV, Bermuda grass white leaf; and 16SrXV Hibiscus witches’- lethal decline (LDN) group; 16SrXXIII, Buckland valley grape- broom. A total of 45 subgroups were identified when ribosomal vine yellows group; 16SrXXIV, Sorghum bunchy shoot group; protein gene RFLP data was also considered in the analyses. 16SrXXV, Weeping tea witches’-broom group; 16SrXXVI, Mau- Sequencing of 30 nearly full-length amplified 16S rRNA ritius sugarcane yellow D3T1 group; 16SrXXVII, Mauritius sug- genes was undertaken by Namba et al. (1993), Gundersen arcane yellow D3T2 group; and 16SrXXVIII, Havana derbid et al. (1994), and Seemüller et al. (1994) from a diversity of phytoplasma group. The virtual RFLP patterns are available strains previously characterized by rDNA RFLP analysis. These for online use as reference patterns at http://www.ba.ars.usda. collective efforts, combined with earlier studies (Kuske and gov/data/mppl/virtualgel.html. Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first The spacer region (SR) separating the 16S from the 23S comprehensive phytoplasma phylogeny. In recognition of their rRNA gene of phytoplasmas was also shown to be a reliable unique phylogenetic status, the trivial name “phytoplasma” was phylogenetic marker. Phylogenetic trees derived from the initially proposed (Sears and Kirkpatrick, 1994) and has since entire 16S–23S SR (Gibb et al., 1998; Kenyon et al., 1998) or been adopted formally (IRPCM Phytoplasma/Spiroplasma variable regions flanking the tRNAile gene (Kirkpatrick et al., Working Team – Phytoplasma Taxonomy Group, 2004) to col- 1995; Schneider et al., 1995) differentiated phytoplasmas into lectively name these fastidious, phytopathogenic mollicutes groups that were concordant with the major groups established previously known as mycoplasma-like organisms. Within the previously from analyses of 16S rRNA genes. Phytoplasmas col- phytoplasma clade, major subclades (primary groups repre- lectively differ in their 16S rRNA gene sequence by no more senting “Candidatus” species) include: (1) Stolbur; (2) Aster than 14%, whereas their respective 16S–23S SR sequences dif- yellows; (3) Apple proliferation; (4) Coconut lethal yellowing; fer by as much as 22%. This added variation has contributed (5) Pigeonpea witches’-broom; (6) X-disease; (7) Rice yellow to improved accuracy of phytoplasma classification at the sub- dwarf; (8) Elm yellows; (9) Ash yellows; (10) Sunnhemp witch- group level. Similarly, phylogenetic analysis of ribosomal pro- es’-broom; (11) Loofah witches’-broom; (12) Clover prolifera- tein genes, secY, secA, or 23S rRNA genes has been employed tion; and (13) Peanut witches’-broom (Kirkpatrick et al., 1995; to differentiate closely related phytoplasma strains, as well as to Schneider et al., 1995; White et al., 1998). Primary phytoplasma aid the group and subgroup classification of diverse phytoplas- groups including 19 novel groups, namely Australian grapevine mas (Daire, 1993; Hodgetts et al., 2008; Lee et al., 1998b, 2004a, yellows (AUSGY), Italian bindweed stolbur (IBS), Buckthorn 2006b; Martini et al., 2007; Reinert, 1999). Such studies have witches’-broom (BWB), Spartium witches’-broom (SpaWB), led to finer differentiation among phytoplasma subgroups and Galactia little leaf (GaLL), Vigna little leaf (ViLL), Clover yellow to enriched descriptions of “Candidatus Phytoplasma” species edge (CYE), Hibiscus witches’-broom (HibWB), Pear decline (Lee et al., 2004a, 2006a, b). (PD), European stone fruit yellows (ESFY), Japanese hydran- A polyphasic system for taxonomy based on integration of gea phyllody (JHP), Psammotettix cephalotes-borne (BVK), Italian genotypic, phenotypic, and phylogenetic information employed alfalfa witches’-broom (IAWB), Cirsium phyllody (CirP), for bacterial classification (Murray et al., 1990; Stackebrandt ­Bermuda grass white leaf (BGWL), Sugarcane white leaf and Goebel, 1994) has proved problematic for nonculturable (SCWL), Tanzanian lethal decline (TLD), Stylosanthes little leaf phytoplasmas. In response to a rapidly growing database of (StLL), and Pinus sylvestris yellows (PinP), that were absent from phylogenetic markers, even in the absence of species-defining previous classification schemes have been subsequently defined. biological or phenotypic characters, the Working Team on Phy- These new taxonomic entities were delineated on the basis of toplasmas of the International Research Programme of Com- phylogenetic tree branching patterns, differences in 16S rRNA parative Mycoplasmology (IRPCM Phytoplasma/Spiroplasma Genus I. “Candidatus Phytoplasma” 703

Working Team – Phytoplasma Taxonomy Group, 2004) pro- separate species. In such cases, the description of two different posed that taxonomy of phytoplasmas be based primarily upon species is recommended when all of the following conditions phylogenetic analyses. This proposal was agreed to and adopted apply: (1) the two phytoplasmas are transmitted by different as policy by the ICSB Subcommittee on the Taxonomy of vector species; (2) the two phytoplasmas have a different natu- Mollicutes­ (1993, 1997), which also recommended that the pro- ral plant host, or at least their symptomatology is significantly visional taxonomic status of “Candidatus”, originally proposed different in the same plant host; (3) there is evidence of signifi- by Murray and Schleifer (1994), be used for assigning genera cant molecular diversity between phytoplasmas as determined names as follows: “Candidatus Phytoplasma” (from phytos, Greek by DNA hybridization assays with cloned nonribosomal DNA for plant; plasma, Greek for thing molded) [(Mollicutes) NC; markers, serological reactions, or by PCR-based assays. The NA; O; NAS (GenBank no. M30790); oligonucleotide sequence taxonomic rank of subspecies should not be used. Reference of unique region of the 16S rRNA gene is CAA GAY BAT KAT strains should be available to the scientific community in graft- GTK TAG CYG GDC T; P (Plant, phloem; Insect, salivary gland); inoculated or in vitro micropropagated host plants or as DNA M]. (IRPCM Phytoplasma/Spiroplasma Working Team –­ if perpetuation of strains in infected host plants is not feasible. Phytoplasma Taxonomy Group, 2004). By this same approach, Descriptions of “Candidatus Phytoplasma” species should be major groups within the genus also delineated by phylogenetic preferably submitted to the International Journal of Systematic analysis of near full-length 16S rRNA gene sequences were con- and Evolutionary Microbiology (http://ijs.sgmjournals.org­ /). sidered to represent one or more distinct species. Recent phylogenetic investigations, including the pres- Current guidelines for “Candidatus Phytoplasma” species ent analyses (Figure 116), suggest 97.5% 16S rRNA gene descriptions (Anonymous, 2000; Firrao et al., 2005; IRPCM sequence similarity may represent a more suitable upper Phytoplasma/Spiroplasma Working Team – Phytoplasma Tax- threshold for “Candidatus Phytoplasma” species separation, onomy Group, 2004) are based upon identification of a signifi- in that taxonomic subgroups designated based on 16S rRNA cantly unique 16S rRNA gene sequence >1200 bp in length. gene sequence similarities of £97.5% more consistently define The strain from which the sequence is obtained should be species that are phylogenetically distinct from nearest related designated as the reference strain. Strains with minimal differ- species. Regardless of homology criteria, a taxonomy is emerg- ences in the 16S rRNA sequence, relative to the reference strain, ing for the phytoplasmas in the absence of cultivability where should be referred to as related strains. In general, a strain can species and related strains of a species are clearly recognized be described as a new “Candidatus Phytoplasma” species if its with due consideration of the genetic, ecological, and environ- 16S rRNA gene sequence has less than 97% identity to any pre- mental constraints unique to this group of plant- and insect- viously described “Candidatus Phytoplasma” species (ICSB Sub- associated Mollicutes. To a large extent, the present taxonomy committee on the Taxonomy of Mollicutes, 2001). There are employs vernacular names based on associated diseases, but cases in which phytoplasmas may share more than 97% of their is constantly shifting towards a traditional taxonomy as more 16S rRNA gene sequence, but clearly represent ecologically and more “Candidatus Phytoplasma” species continue to be distinct populations and, thus, they may warrant description as ­recognized and proposed.

List of species of the genus “Candidatus Phytoplasma” In accordance with the current guidelines for “Candidatus Morphology: other. ­Phytoplasma” species descriptions, the following species have Sequence accession no. (16S rRNA gene): DQ174122. been designated. Proposed assignments to the class Mollicutes Unique regions of 16S rRNA gene: 5¢-GTTTCTTCGGAAA-3¢ are based on nucleic acid sequences. None of these species have (68–80), 5¢-GTTAGAAATGACT-3¢ (142–153), 5¢-GCTGGT- been cultivated independently of their host, and their metabo- GGCTT-3¢ (1438–1448). lism and growth temperatures are unknown. Habitat, association, or host: Solanum tuberosum phloem. 1. “Candidatus Phytoplasma allocasuarinae” Marcone, Gibb, 3. “Candidatus Phytoplasma asteris” Lee, Gundersen-Rindal, Streten and Schneider 2004a, 1028 Davis, Bottner, Marcone and Seemüller 2004a, 1046 Vernacular epithet: Allocasuarina yellows phytoplasma, Vernacular epithet: Aster yellows (AY) phytoplasma, strain strain AlloYR. OAY R. Gram reaction: not applicable. Gram reaction: not applicable. Morphology: other. Morphology: other. Sequence accession no. (16S rRNA gene): AY135523. Sequence accession no. (16S rRNA gene): M30790. Unique region of 16S rRNA gene: 5¢-TTTATTCGAGAG- Unique regions of 16S rRNA gene: 5¢-GGGAGGA-3¢, GGCG-3¢. 5¢-CTGACGGTACC-3¢, and 5¢-CACAGTGGAGGTTAT- Habitat, association, or host: phloem of Allocasuarina muel- CAGTTG-3¢. leriana (Slaty she-oak). Habitat, association, or host: phloem of Oenothera hookeri (Evening primrose). 2. “Candidatus Phytoplasma americanum” Lee, Bottner, Secor and Rivera-Varas 2006a, 1596 4. “Candidatus Phytoplasma aurantifolia” Zreik, Carle, Bové Vernacular epithet: Potato purple top, strain APPTW12- and Garnier 1995, 452 NER. Vernacular epithet: Witches’-broom disease of lime phyto- Gram reaction: not applicable. plasma, strain WBDLR. 704 Family II. Incertae sedis

77 P.trifoli (CP) 79 P. fraxini (AshY1) 100 P. ulmi (EY1) 100 P. ziziphi (JWB-G1) 93 LfWB StLL 56 LDG LDT LY-c2 72 SBS 88 P. castaneae (CnWB) b P. pini (PinP) CIRP GaLL 98 BVK 100 P. oryzae (RYD) SCWL 98 P. cynodontis (BGWL) 100 P. phoenecium (AlmWB-A4) ViLL WX

59 GLL-eth P. brasiliense (HibWB) IAWB SPLL 100 P. australasia (PpYC) 91 P. aurantifolia (WBDL) WTWB P. mali (AP15) P. prunorum (ESFY-G1) P. pyri (PD1) a 100 P. spartii (SPAR) P. allocasuarinae (AlloY) P. rhamni (BWB) STOL 100 P. australiense (AusGY) P. japonicum (JHP) IBS 100 P. asteris (MIAY) MPV A. palmae A. laidlawii 10 changes

FIGURE 116. Phylogenetic analysis of the phytoplasmas. Phylogenetic trees were constructed by parsimony analy- ses of phytoplasma 16S rRNA gene sequences using the computer program PAUP (Swofford, 1998). The closely related culturable Acholeplasma palmae was employed as the outgroup. Because phytoplasma taxa are too numerous to present in a single inclusive tree, a global phylogeny of representative phytoplasmas is first presented. The global tree is divided into lower (a) and upper (b) regions. Each region of the global phylogeny is then expanded into inclusive trees, a and b, which collectively include 145 phytoplasmas from diverse geographic origins. Taxonomic subgroups, representing phytoplasmas sharing at least 97.5% 16S rRNA gene sequence similarity, are identified on each inclusive tree. Each phylogenetically distinct subgroup is equivalent to a subclade (or putative species) within the genus “Candidatus Phytoplasma”. In all trees, branch lengths are proportional to the number of inferred char- acter state transformations. Bootstrap (confidence) values greater than or equal to 50 are shown on the branches. Phytoplasmas for which 16S rRNA gene sequences of at least 1200 bp in length have been determined (312 total) are listed by subgroup in Table 144 along with their sequence accession numbers. Genus I. “Candidatus Phytoplasma” 705

SUNHP a SPWB PnWB AlWB P. australasia GPh P. australasia (PpYC) CoAWB IAWB IAWB 84 PEP SPLL SPLL GLL-eth GLL-eth 100 P. brasiliense (HibWB) P. brasiliense CaM CaWB1 100 87 FBP P. aurantifolia P. aurantifolia (WBDL) BoLL BoLL WTWB P. mali (AP15) WTWB AP1/93 AP2 P. mali 52 AT P. pyri (PD1) P. pyri 100 PD PPER P. prunorum (ESFY-G1) 82 ESFY-142 P. prunorum ESFY4 100 ESFY5 P. spartii (Spar) P. spartii P. allocasuarinae (AlloY) 100 P. allocasuarinae P. rhamni (BWB) P. asteris (MIAY) P. rhamni PPT MBS PaWB MD Bstv2M.f12 HyPH APWB IOWB PRIVC RPh ApSL AAY P. asteris SAY ValY WcWB OY-M CabD3 AY-WB 100 BB HYDP CPh STRAWB2 BBS ACLR CWL SY PY STOL 100 100 VK STOL 80 STOL2 PpDB PYL 65 SLY P. australiense SV3101 95 P. australiense (AusGY) P. japonicum (JHP) P. japonicum 51 IBS IBS MPV 100 STRAWB1 MPV CbY1 A. palmae 5 changes

FIGURE 116. (Continued)

Gram reaction: not applicable. Habitat, association, or host: phloem of Carica papaya and Morphology: other. Lycopersicon esculentum. Sequence accession no. (16S rRNA gene): U15442. 6. “Candidatus Phytoplasma australiense” Davis, Dally, Gun- Unique region of 16S rRNA gene: 5¢-GCAAGTGGTGAAC- dersen, Lee and Habili 1997, 268 CATTTGTTT-3¢. Vernacular epithet: Australian grapevine yellows phyto- Habitat, association, or host: phloem of Citrus; hemolymph R plasma, strain AUSGY . and salivary glands of Hishimonus phycitis (Cicadellidae). Gram reaction: not applicable. 5. “Candidatus Phytoplasma australasia” White, Blackall, Scott Morphology: other. and Walsh 1998, 949 Sequence accession no. (16S rRNA gene): L76865. Vernacular epithet: Papaya yellow crinkle phytoplasma, Unique regions of 16S rRNA gene: 5¢-CGGTAGAAATAT­ strain PpYCR. CGT-3¢ and 5¢-TTTATCTTTAAAAGACCTCGCAAGA-3¢. Gram reaction: not applicable. Habitat, association, or host: Vitis phloem. Morphology: other. 7. “Candidatus Phytoplasma brasiliense” Montano, Davis, Sequence accession no. (16S rRNA gene): Y10097. ­Dally, Hogenhout, Pimentel and Brioso 2001, 1117 Unique regions of 16S rRNA gene: 5¢-TAAAAGGCATCTTT- Vernacular epithet: Hibiscus witches’-broom (HibWB) TATC-3¢ and 5¢-CAAGGAAGAAAAGCAAATGGCGAAC- ­phytoplasma, strain HibWB26R. CATTTGTTT-3¢. 706 Family II. Incertae sedis

b P. trifolii (CP) PWB BLL-bd 100 BLL P. trifolii CSV BLTVA 88 VR ArAWB EriWB EriWB 100 P. fraxini (AshY1) ASHY P. fraxini LWB3 AshY3 86 ALY FD HD1 SpaWB229 P. ulmi VC RuS P. ulmi (EY1) 100 100 ULW JWB-ch P. ziziphi (JWB-G1) CLY-5 P. ziziphi NecY-In1 JWB-Ka LfWB LfWB 100 LfWB-t StLL StLL CY LY-c2 PanD LY ScY LDY 81 LfY1 56 LDG 72 LDG LDN LDT LDT SBS SBS SGS-v1 SCWL SCWL BVK BVK 100 CIRP CIRP 51 GaLL GaLL P. oryzae (RYD-J) RYD-Th P. oryzae BGWL-2 CWL P. cynodontis P. cynodontis (BGWL-C1) 97 BGWL 88 P. casteneae (CnWB) P. casteneae P. pini (Pin127) P. pini PinG KAP P. phoenecium 100 PPWB-f P. phoenecium (AlmWB-A4) ViLL ViLL BBP TWB VAC CYE-C DanVir-a GDIII CbY18 WWB-a WX BLWB 100 PoiBI VGYIII CX ScYPI-Afr WX LP A. palmae 5 changes

FIGURE 116. (Continued)

Gram reaction: not applicable. 8. “Candidatus Phytoplasma caricae” Arocha, López, Piñol, Morphology: other. Fernández, Picornell, Almeida, Palenzuela, Wilson and Sequence accession no. (16S rRNA gene): AF147708. Jones 2005, 2462 Unique regions of 16S rRNA gene: 5¢-GAAAAAGAAAG-3¢, Vernacular epithet: Cuban papaya phytoplasma, strain PAYR. 5¢-TCTTTCTTT-3¢, 5¢-CAG-3¢, 5¢-ACTTTG-3¢, and 5¢-GTCA Gram reaction: not applicable. AAAC-3¢. Morphology: other. Habitat, association, or host: Hibiscus phloem. Sequence accession no. (16S rRNA gene): AY725234. Genus I. “Candidatus Phytoplasma” 707

Table 144. Provisional groupings, strain designations, associated plant disease, geographic origin and accession numbers of 16S rRNA gene sequences derived from phytoplasmasa

“Candidatus Subgroup Strain Associated plant disease Phytoplasma species” Geographic origin 16S accession no.a AlloY AlloY Allocasuarina yellows P. allocasuarinae Australia AY135523* AP AP15R Apple proliferation P. mali Italy AJ542541* AT Apple proliferation Germany X68375* AP2 Apple proliferation Germany AF248958* AP1/93 Apple proliferation France AJ542542* D365/04 Apple proliferation Slovenia EF025917 APSb Apple proliferation Italy EF193361 ESFY ESFY-G1R European stone fruit yellows P. prunorum Germany AJ542544* ESFY5 European stone fruit yellows Austria AY029540* ESFY4 European stone fruit yellows Czech Republic Y11933* PPER European stone fruit yellows Germany X68374 ESFY-142 European stone fruit yellows Spain AJ575108* ESFY-173 European stone fruit yellows Spain AJ575106 ESFY-215 European stone fruit yellows Spain AJ575105 AshY AshY1 Ash yellows P. fraxini USA, New York AF092209* AshY3 Ash yellows USA, Utah AF105315* ASHY Ash yellows Germany X68339* LWB3 Lilac witches’-broom USA, Massachusetts AF105317* EriWB EriWB Erigeron witches’-broom Brazil AY034608 ArAWB Argentinian alfalfa witches’- Argentina AY147038 broom AusGY AusGY Australian grapevine yellows P. australiense Australia L76865 PpDB Papaya die-back Australia Y10095* PYL Phormium yellow leaf, rrnA New Zealand U43569 PYLb Phormium yellow leaf New Zealand U43570* SLY Strawberry lethal yellows Australia AJ243045* SV3101 Strawberry virescence Tonga AY377868* AY MIAY Oenothera virescence P. asteris USA, Michigan M30790* OY-M Onion yellows Japan NC005303* MBS Maize bushy stunt Mexico AY265208* APWB Aphanamixis polystachya Bangladesh AY495702* witches’-broom IOWB Ipomoea obscura witches’- Taiwan AY265205* broom HyPH Hydrangea phyllody Italy AY265207* HYPh Hydrangea phyllody France AY265219 RPh Oilseed rape phyllody Czech Republic U89378* MD Mulberry dwarf South Korea AY075038* GDS DQ112021 AYWB_ro4 Aster yellows witches’- Ohio, USA NC007716 broom PaWB Paulownia witches’-broom Korea AF279271* PY1 Periwinkle yellows China AF453328 BVGY AY083605 AAY American aster yellows Southern USA X68373* CabD4 Cabbage proliferation USA, Texas AY180932 AY-BW Aster yellows USA, Ohio AY389820 ApSL Apple sessile leaf Lithuania AY734454 SAY Severe aster yellows USA, California M86340* ValY Valeriana yellows, rrnA Lithuania AY102274* PRIVC Primrose virescence Germany AY265210* WcWB Watercress witches’-broom USA, Hawaii AY665676* CabD3 Cabbage proliferation USA, Texas AY180947* AY-sb Sugar beet aster yellows Hungary AF245439 Bstv2Mf12 AY180951 ACLR-AY Apricot chlorotic leafroll Spain AY265211 ACLR Apricot chlorotic leafroll Europe X68338* BBS3 Blueberry stunt USA, Michigan AY265213 STRAWB2 Strawberry green petal USA, Florida U96616* PoY Populus yellows Croatia AF503568 KVG Clover phyllody Germany X83870

(continued) 708 Family II. Incertae sedis

Table 144. (continued) “Candidatus Subgroup Strain Associated plant disease Phytoplasma species” Geographic origin 16S accession no.a CPh Clover phyllody Canada AF222066* HYDP Hydrangea phyllody Belgium AY265215* AY-WB Aster yellows USA, Ohio AY389827* BB Tomato big bud USA, Arkansas AY180955* PPT Potato purple top Mexico AF217247* THP THP Tomato ‘hoja de perejil’ P. lycopersici Bolivia AY787136 Derbid Derbid phytoplasma Cuba AY744945 BWB BWB Buckthorn witches’-broom P. rhamni Germany X76431* BGWL BGWL-C1 Bermudagrass white leaf P. cynodontis Italy AJ550984* BGWL Bermudagrass white leaf Italy Y16388* BGWL-2 Bermudagrass white leaf Thailand AF248961* CWL Cynodon white leaf Australia AF509321* BVK BVK Psammotettic cephalotes- Germany X76429* borne CIRP CIRP Cirsium phyllody Germany X83438* CnWB CnWB Chestnut witches’- broom P. castaneae Korea AB054986* CP CPR Clover proliferation P. trifolii Canada AY390261* BLL Brinjal little leaf India X83431* BLTVA Columbia basin potato USA, Washington AY692280* purple top VR Vinca virescence USA, California AY500817* PWB Potato witches’-broom Canada AY500818* CSV Centauria stolstitialis Italy AY270156* virescence EY EY1 Elm yellows P. ulmi USA, New York AY197655* ULW Ulmus witches’-broom Italy X68376* FD Flavescence doree Italy X76560* RuS Rubus stunt Italy AY197648* HD1 Hemp dogbane yellows USA, New York AY197654* VC Asymptomatic Virginia USA, Florida AF305198* creeper SpaWB SpaWB229 Spartium witches’-broom P. spartii Italy AY197652* JWB JWB-G1T Jujube witches’-broom Gifu P. ziziphi Japan AB052876* isolate 1 JWB-Ka Jujube witches’-broom Korea AB052879* Korea isolate 1 JWB-ch Ziziphus jujube witches’- China AF305240 broom NecY-In1 Nectarine yellows India AY332659* CLY-5 Cherry lethal yellows China AY197659* FBP WBDL Witches’-broom disease of P. aurantifolia Oman U15442* lime CaWB-YNO1 Cactus witches’-broom China AJ293216 FBP Faba bean phyllody Sudan X83432* PPLL Pigeon pea little leaf Australia AJ289191 BoLL BoLL Bonamia little leaf Australia Y15863* GaLL GaLL Galactia little leaf Australia Y15865* GLL-eth GLL-eth Gliricidia little leaf Ethiopia AF361018* HibWB HibWB Hibiscus witches’-broom P. brasiliense Brazil AF147708* IAWB IAWB Alfalfa witches’-broom Italy Y16390* PEP Pichris echioides phyllody Italy Y16393* IBS IBS Italian bindweed yellows Southern Italy Y16391* StrawY StrawY Strawberry lethal yellows P. fragariae Lithuania DQ086423 JHP JHP Japanese hydrangea P. japonicum Japan AB010425 phyllody LDG LDG Cape St Paul wilt Ghana Y13912* LDN Awka disease of coconut Nigeria Y14175* LDT LDT Coconut lethal disease Tanzania X80177* LfWB LfWB Loofah WB Taiwan L33764* LfWB-t Loofah WB Taiwan AF086621* LY CPY Carludovica palmata yellows Mexico AF237615 LDY Yucatan coconut decline Mexico U18753*

(continued) Genus I. “Candidatus Phytoplasma” 709

Table 144. (continued) “Candidatus Subgroup Strain Associated plant disease Phytoplasma species” Geographic origin 16S accession no.a LfY1 Coconut leaf yellowing Mexico AF500329* LfY5(PE65) Coconut leaf yellowing Mexico AF500334 LY-c2 Coconut lethal yellows USA, Florida AF498309* LY-JC8 Coconut lethal yellows Jamaica AF498307 PanD Pandanus decline USA, Florida AF361020* ScY Sugarcane yellows, group 4 Mauritius AJ539178* ScY SCD3T2 Sugarcane yellows, group 3 Mauritius AJ539180 SCD3T1 Sugarcane yellows, group 3 Mauritius AJ539179 MPV MPV Mexican periwinkle Mexico AF248960* virescence PerWB-FL Periwinkle witches’-broom USA, Florida AY204549 CbY1 Chinaberry yellows Bolivia AF495882* STRAWB1 Strawberry green petal USA, Florida U96614* PD PD1 Pear decline P. pyri Italy AJ542543* PD Pear decline Germany X76425* PYLR Peach yellow leafroll USA, California Y16394 EPC Pear decline Iran DQ471321 PinP Pin127R Pinus halepensis yellows P. pini Spain AJ632155* PinG Pinus sylvestris yellows Germany AJ310849* PPWB AlmWB-A4 Almond witches’-broom P. phoenecium Lebanon AF515636* KAP Knautia arvensis phyllody Italy Y18052* PPWB-f Pigeonpea witches’-broom USA, Florida AF248957* RYD RYD-J Rice yellow dwarf P. oryzae Japan D12581 RYD-Th Rice yellow dwarf Thailand AB052873* SBS SBS Sorghum bunchy shoot Australia AF509322* SCWL SCWL Sugarcane white leaf Thailand X76432* SGS-v1 Sorghum grassy shoot, Australia AF509324* variant 1 SpaWB Spar Spartium witches’-broom P. spartii Italy X92869* SPLL SPLL Sweet potato little leaf Australia X90591* SPWB PpYC Papaya yellow crinkle P. australasia Australia Y10097* GPh Gerbera phyllody Japan? AB026155* PnWB Peanut witches’ broom Taiwan L33765* CoAWB Cocky apple witches’-broom Australia AJ295330* SPLL Sweet potato little leaf Australia AJ289193 SUNHP Sunnhemp phyllody Thailand X76433* AlWB Alfalfa witches’-broom Oman AY169322* TBB Australian tomato big bud Australia Y08173 StLL StLL Stylosanthes little leaf Australia AJ289192* STOL STOL Stolbur of Capsicum annum Europe X76427* VK Grapevine yellows Europe X76428* 2642BN Grapevine yellows P. solani France AJ964960 ViLL ViLL Vigna little leaf Australia Y15866* CIWB IM-3 Cassia italica witches’-broom P. omaniense Oman EF666051 WTWB WTWB Weeping tea witches’-broom Australia AF521672* WX BBP Blueberry proliferation Lithuania AY034090* BLWB Black locust witches’-broom USA, Maryland AF244363* CbY18 Chinaberry yellows Bolivia AF495657* CX Canadian peach X Canada L33733* CYE Clover yellow edge Canada AF175304* DanVir-a Dandelion virescence, rrnA Lithuania AF370119* LP Little peach USA, S. Carolina AF236122* PoiBI Poinsettia branch-inducing Southern USA AF190223* ScYP I-Afr Sugarcane yellows Africa AF056095* TWB Tsuwabuki WB Japan D12580* VAC Vaccinium witches’-broom Germany X76430* VGYIII Virginia grapevine yellows USA, Virginia AF060875* WWB-a Walnut witches’-broom, USA, Georgia AF190226* rrnA WX Western X USA, California L04682* aAccession numbers denoted by an asterisk were used as sources of 16S rRNA gene sequences for comprehensive phylogenetic analysis of subgroup phytoplasmas from .diverse geographic origins 710 Family II. Incertae sedis

Unique regions of 16S rRNA gene: 5¢-AAA-3¢ (196–198), 5¢- (1381–1383), and 5¢-ATTTACGTTTCTG-3¢ (1392–1404). ATT-3¢ (600–603), 5¢-AGGCGCC-3¢ (1089–1095), 5¢-GCG- Habitat, association, or host: Saccharum officinarum phloem. GATTTAGTCACTTTTCAGGC-3¢ (1379–1401). 14. “Candidatus Phytoplasma japonicum” Sawayanagi, Habitat, association, or host: Carica papaya phloem. ­Horikoshi, Kanehira, Shinohara, Bertaccini, Cousin, Hiruki 9. “Candidatus Phytoplasma castaneae” Jung, Sawayanagi, and Namba 1999, 1284 Kakizawa, Nishigawa, Miyata, Oshima, Ugaki, Lee, Hibi and Vernacular epithet: Japanese Hydrangea phyllody phyto- Namba 2002, 1548 plasma, strain JHPR. Vernacular epithet: Chestnut witches’ broom phytoplasma, Gram reaction: not applicable. strain CnWBR. Morphology: other. Gram reaction: not applicable. Sequence accession no. (16S rRNA gene): AB010425. Morphology: other. Unique regions of 16S rRNA gene: 5¢-GTGTAGCCG- Sequence accession no. (16S rRNA gene): AB054986. GGCTGAGAGGTCA-3¢ and 5¢-TCCAACTCTAGCTAAA- Unique regions of 16S rRNA gene: 5¢-CTAGTTTAAAAA- CAGTTTCTG-3¢. CAATGCTC-3¢ and 5¢-CTCATCTTCCTCCAATTC-3¢. Habitat, association, or host: Hydrangea phloem. Habitat, association, or host: Castanea crenata phloem. 15. “Candidatus Phytoplasma lycopersici” Arocha, Antesana, 10. “Candidatus Phytoplasma cynodontis” Marcone, Schneider Montellano, Franco, Plata and Jones 2007, 1709 and Seemüller 2004b, 1081 Vernacular epithet: Tomato “hoja de perejil” phytoplasma, Vernacular epithet: Bermuda grass white leaf (BGWL) phy- strain THPR. toplasma, strain BGWl-C1R. Gram reaction: not applicable. Gram reaction: not applicable. Morphology: other. Morphology: other. Sequence accession no. (16S rRNA gene): AY787136. Sequence accession no. (16S rRNA gene): AJ550984. Unique regions of 16S rRNA gene: 5¢-CTTA-3¢ (positions Unique region of 16S rRNA gene: 5¢-AATTAGAAGGCAT­ 175–178), 5¢-AATGGT-3¢ (198–203), 5¢-ATA-3¢ (229–231), CTTTTAAT-3¢. 5¢-TGGAGGAA-3¢ (234–242), 5¢-CACG-3¢ (302–305), Habitat, association, or host: phloem of Cynodon dactylon 5¢-TCT-3¢ (315–317), 5¢-GCT-3¢ (334–336), 5¢-TAT-3¢ (Bermuda grass). (336–338), 5¢-TAC-3¢ (413–415), and 5¢-AGC-3¢ (434–436). 11. “Candidatus Phytoplasma fragariae” Valiunas, Staniulis and Habitat, association, or host: Lycopersicon esculentum Davis 2006, 280 phloem. Vernacular epithet: Strawberry yellows phytoplasma, strain 16. “Candidatus Phytoplasma mali” Seemüller and Schneider StrawY R. 2004, 1224 Gram reaction: not applicable. Vernacular epithet: Apple proliferation (AP) phytoplasma, Morphology: other. strain AP15R. Sequence accession no. (16S rRNA gene): DQ086423. Gram reaction: not applicable. Unique regions of 16S rRNA gene: 5¢-GTGCAATGCT- Morphology: other. CAACGTTGTGAT-3¢, 5¢-AATTGCA-3¢, and 5¢-TGAGTAAT- Sequence accession no. (16S rRNA gene): AJ542541. CAAGAGGGAG-3¢. Unique region of 16S rRNA gene: 5¢-AATACTCGAAACCA- Habitat, association, or host: phloem of Fragaria x ananassa. GTA-3¢. 12. “Candidatus Phytoplasma fraxini” Griffiths, Sinclair, Smart Habitat, association, or host: Malus phloem. and Davis 1999, 1613 17. “Candidatus Phytoplasma omanense” Al-Saady, Khan, Vernacular epithet: Ash yellows phytoplasma, strain AshYR ­Calari, Al-Subhi and Bertaccini 2008, 464 and lilac witches’-broom (LWB) phytoplasma. Vernacular epithet: Cassia witches’-broom (CWB) phyto- Gram reaction: not applicable. plasma, strain IM-1R. Morphology: other. Gram reaction: not applicable. Sequence accession no. (16S rRNA gene): AF092209. Morphology: other. Unique regions of 16S rRNA gene: 5¢-CGGAAACCCCT- Sequence accession no. (16S rRNA gene): EF666051. CAAAAGGTTT-3¢ and 5¢-AGGAAAGTC-3¢. Unique regions of 16S rRNA gene: 5¢-AAAAAACAGT-3¢ (467– Habitat, association, or host: phloem of Fraxinus and 474), 5¢-TTGC-3¢ (642–645), 5¢-GTTAAAG-3¢ (853–861), Syringa. 5¢-TAATT-3¢ (1010–1014), and 5¢-AAATT-3¢ (1052–1056). 13. “Candidatus Phytoplasma graminis” Arocha, López, Piñol, Habitat, association, or host: Cassia italica phloem. Fernández, Picornell, Almeida, Palenzuela, Wilson and 18. “Candidatus Phytoplasma oryzae” Jung, Sawayanagi, Wong- Jones 2005, 2462 kaew, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Vernacular epithet: Sugarcane yellow leaf phytoplasma, Hibi and Namba 2003c, 1928 strain SCYPR. Vernacular epithet: Rice yellow dwarf (RYD) phytoplasma, Gram reaction: not applicable. strain RYD-ThR. Morphology: other. Gram reaction: not applicable. Sequence accession no. (16S rRNA gene): AY725228. Morphology: other. Unique regions of 16S rRNA gene: 5¢-TTTG-3¢ (465–468), Sequence accession nos (16S rRNA gene): D12581, AB052873 5¢-TTG-3¢ (478–480), 5¢-GGG-3¢ (1552–1554), 5¢-TAA-3¢ (RYD-Th). Genus I. “Candidatus Phytoplasma” 711

Unique regions of 16S rRNA gene: 5¢-AACTGGATAGGAAAT- Vernacular epithet: Stolbur phytoplasma; subgroup A ref- TAAAAGGT-3¢ and 5¢-ATGAGACTGCCAATA-3¢. erence type of the stolbur phytoplasma taxonomic group Habitat, association, or host: Oryza sativa phloem. 16SrXII (Lee et al., 2000). Gram reaction: not applicable. 19. “Candidatus Phytoplasma phoenicium” Verdin, Salar, Danet, Morphology: other. Choueiri, Jreijiri, El Zammar, Gélie, Bové and Garnier 2003, 837 Sequence accession no. (16S rRNA gene): AJ970609 (strain Vernacular epithet: Almond witches’-broom (AlmWB) PO; Cimerman et al., 2006). phyto­plasma, strain AlmWB-A4R. Unique region of 16S rRNA gene: not reported. Gram reaction: not applicable. Habitat, association, or host: many species of Solanaceae plus Morphology: other. several species in other plant families, and Fulguromorpha Sequence accession no. (16S rRNA gene): AF515636. spp. planthopper vectors. Unique region of 16S rRNA gene: 5¢-CCTTTTTCGGAAGG- TATG-3¢. 25. “Candidatus Phytoplasma spartii” Marcone, Gibb, Streten Habitat, association, or host: Prunus amygdalus phloem. and Schneider 2004a, 1028 Vernacular epithet: Spartium witches’-broom phytoplasma, 20. “Candidatus Phytoplasma pini” Schneider, Torres, Martín, strain SpaWBR. Schröder, Behnke and Seemüller 2005, 306 Gram reaction: not applicable. Vernacular epithet: Pinus halepensis yellows (Pin) phyto- Morphology: other. plasma, strain Pin127SR. Sequence accession no. (16S rRNA gene): X92869. Gram reaction: not applicable. Unique region of 16S rRNA gene: 5¢-TTATCCGCGTTAC-3¢. Morphology: other. Habitat, association, or host: phloem of Spartium junceum Sequence accession no. (16S rRNA gene): AJ632155. (Spanish broom). Unique regions of 16S rRNA gene: 5¢-GGAAATCTTTCG- GGATTTTAGT-3¢ and 5¢-TCTCAGTGCTTAACGCTGT- 26. “Candidatus Phytoplasma tamaricis” Zhao, Sun, Wei, Davis, TCT-3¢. Wu and Liu 2009, 2496 Habitat, association, or host: Pinus phloem. Vernacular epithet: Salt cedar witches’-broom phytoplasma, strain SCWB1R. 21. “Candidatus Phytoplasma prunorum” Seemüller and Sch- Gram reaction: not applicable. neider 2004, 1224 Morphology: other. Vernacular epithet: European stone fruit yellows (ESFY) Sequence accession no. (16S rRNA gene): FJ432664. phytoplasma, strain ESFY-G1R. Unique regions of 16S rRNA gene: 5¢-ATTAGGCATCTAG- Gram reaction: not applicable. TAACTTTG-3¢, 5¢-TGCTCAACATTGTTGC-3¢, 5¢-AGCTTT- Morphology: other. GCAAAGTTG-3¢, and 5¢-TAACAGAGGTTATCAGAGTT-3¢. Sequence accession no. (16S rRNA gene): AJ542544. Habitat, association, or host: phloem of Tamarix chinensis Unique regions of 16S rRNA gene: 5¢-AATACCCGAAACCA- (salt cedar). GTA-3¢ and 5¢-TGAAGTTTTGAGGCATCTCGAA-3¢. Habitat, association, or host: Prunus phloem. 27. “Candidatus Phytoplasma trifolii” Hiruki and Wang 2004, 1352 Vernacular epithet: Clover proliferation phytoplasma, 22. “Candidatus Phytoplasma pyri” Seemüller and Schneider strain CPR. 2004, 1224 Gram reaction: not applicable. Vernacular epithet: Pear decline (PD) phytoplasma, strain Morphology: other. PD1R. Sequence accession no. (16S rRNA gene): AY390261. Gram reaction: not applicable. Unique regions of 16S rRNA gene: 5¢-TTCTTACGA-3¢ and Morphology: other. 5¢-TAGAGTTAAAAGCC-3¢. Sequence accession no. (16S rRNA gene): AJ542543. Habitat, association, or host: Trifolium phloem. Unique regions of 16S rRNA gene: 5¢-AATACTCAAAACCA- GTA-3¢ and 5¢-ATACGGCCCAAACTCATACGGA-3¢. 28. “Candidatus Phytoplasma ulmi” Lee, Martini, Marcone and Habitat, association, or host: Pyrus phloem. Zhu 2004b, 345 Vernacular epithet: Elm yellows phytoplasma (EY) phyto- 23. “Candidatus Phytoplasma rhamni” Marcone, Gibb, Streten plasma, strain EY1R. and Schneider 2004a, 1028 Gram reaction: not applicable. Vernacular epithet: Buckthorn witches’-broom phyto- Morphology: other. plasma, strain BWBR. Sequence accession nos (16S rRNA gene): AY197655, Gram reaction: not applicable. AY197675, and AY197690. Morphology: other. Unique regions of 16S rRNA gene: 5¢-GGAAA-3¢ and 5¢-CGT- Sequence accession nos (16S rRNA gene): X76431, AJ583009. TAGTTGCC-3¢. Unique regions of 16S rRNA gene: 5¢-CGAAGTATTTCGA- Habitat, association, or host: Ulmus americana phloem. TAC-3¢. Habitat, association, or host: phloem of Rhamnus catharticus 29. “Candidatus Phytoplasma vitis” Firrao, Gibb and Streton (buckthorn). 2005, 251 Vernacular epithet: Flavescence dorée phytoplasma; strains 24. “Candidatus Phytoplasma solani” Firrao, Gibb and Streton are genetically heterogenous and vary in degree of virulence, 2005, 251 but all are referable to subgroups C or D of the elm yellows 712 Family II. Incertae sedis

phytoplasma taxonomic group 16SrV (Lee et al., 2000). Vernacular epithet: Jujube witches’-broom phytoplasma, Gram reaction: not applicable. strain JWBR. Morphology: other. Gram reaction: not applicable. Sequence accession nos (16S rRNA gene): AY197645 (16SrV Morphology: other. subgroup C), AY197644 (16SrV subgroup D1). Sequence accession nos (16S rRNA gene):AB052875– Unique regions of 16S rRNA gene: not reported. AB052879. Habitat, association, or host: grapevines (Vitis vinifera) and Unique regions of 16S rRNA gene: 5¢-TAAAAAGGCATCTT­ the leafhopper vector Scaphoideus titanus. TTTGTT-3¢ and 5¢-AATCCGGACTAAGACTGT-3¢. Habitat, association, or host: Ziziphyus jujube phloem. 30. “Candidatus Phytoplasma ziziphi” Jung, Sawayanagi, Kak- izawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi and Namba 2003b, 1041

References highly divergent in the major hydrophilic region. Microbiology 148: 157–167. Ahrens, U. and E. Seemüller. 1992. Detection of DNA of plant patho- Baric, S. and J. Dalla-Via. 2004. A new approach to apple proliferation genic mycoplasma-like organisms by a polymerase chain reaction detection: a highly sensitive real-time PCR assay. J. Microbiol. Meth- that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: ods 57: 135–145. 828–832. Berg, M., D.L. Davies, M.F. Clark, H.J. Vetten, G. Maier, C. Marcone and Al-Saady, N.A., A.J. Khan, A. Calari, A.M. Al-Subhi and A. Bertaccini. E. Seemüller. 1999. Isolation of the gene encoding an immunodomi- 2008. ‘Candidatus Phytoplasma omanense’, associated with witches’- nant membrane protein of the apple proliferation phytoplasma and broom of Cassia italica (Mill.) Spreng. in Oman. Int. J. Syst. Evol. expression and characterization of the gene product. Microbiology Microbiol. 58: 461–466. 145: 1937–1943. Albertazzi, G., A.C. J. Milc, E. Francia, E. Roncaglia, F. Ferrari, E. Taglia- Berg, M. and E. Seemüller. 1999. Chromosomal organization and nucle- fico, E. Stefani and N. Pecchioni. 2009. Gene expression in grapevine otide sequence of the genes coding for the elongation factors G and cultivars in response to Bois Noir phytoplasma infection. Plant Sci- Tu of the apple proliferation phytoplasma. Gene 226: 103–109. ence 176: 792–804. Bertaccini, A., R.E. Davis, R.W. Hammond, M. Vibio, M.G. Bellardi and Alma, A., D. Bosco, A. Danielli, A. Bertaccini, M. Vibio and A. Arzone. I.-M. Lee. 1992. Sensitive detection of mycoplasmalike organisms in 1997. Identification of phytoplasmas in eggs, nymphs and adults of field-collected and in vitro propagated plants of Brassica, Hydrangea Scaphoideus titanus Ball reared on healthy plants. Insect. Mol. Biol. 6: and Chrysanthemum by polymerase chain reaction. Ann. Appl. Biol. 115–121. 121: 593–599. An, F.-Q., Y.-F. Wu, X.-Q. Sun, P.-W. Gu and Y. Yang. 2006. Homologic Bertaccini, A., J. Fráova, S. Paltrinieri, M. Martini, M. Navrátil, C. Luga- analysis of tuf gene for elongation factor Tu of phytoplasma from resi, Nebesárová and M. Simkova. 1999. Leek proliferation: a new wheat blue dwarf. Sci. Agric. Sinica 39: 74–80. phytoplasma disease in the Czech Republic and Italy. Eur. J. Plant Anonymous. 2000. Report of consultations: Phytoplasmas, spiroplas- Pathol. 105: 487–493. mas, mesoplasmas and entomoplasmas working team. Presented at Bertamini, M. and N. Nedunchezhian. 2001. Effects of phytoplasma the International Research Programme on Comparative Mycoplas- [stolbur-subgroup­ (Bois noir-BN)] on photosynthetic pigments, sac- mology (IRPCM) of the International Organization for Mycoplas- charides, ribulose 1,5-biphosphate carboxylase, nitrate and nitrite mology (IOM) Fukuoka, Japan. reductases, and photosynthetic activities in field-grown grapevine (Vitis Arashida, R., S. Kakizawa, Y. Ishii, A. Hoshi, H.Y. Jung, S. Kagiwada, vinifera L. cv. Chardonnay) leaves. Photosynthetica 39: 119–122. Y. Yamaji, K. Oshima and S. Namba. 2008. Cloning and character- Bianco, P.A., R.E. Davis, J.P. Prince, I.-M. Lee, D.E. Gundersen, A. For- ization of the antigenic membrane protein (Amp) gene and in situ tusini and G. Belli. 1993. Double and single infections by aster yellows detection of Amp from malformed flowers infected with Japanese and elm yellows MLOs in grapevines and symptoms characteristic of hydrangea phyllody phytoplasma. Phytopathology 98: 769–775. Flavescence dorée. Rev. Patol. Veg. 3: 69–82. Arocha, Y., M. Lopez, B. Pinol, M. Fernandez, B. Picornell, R. Almeida, Blomquist, C.L., D.J. Barbara, D.L. Davies, M.F. Clark and B.C. Kirk- I. Palenzuela, M.R. Wilson and P. Jones. 2005. ‘Candidatus Phyto- patrick. 2001. An immunodominant membrane protein gene from plasma graminis’ and ‘Candidatus Phytoplasma caricae’, two novel the Western X-disease phytoplasma is distinct from those of other phytoplasmas associated with diseases of sugarcane, weeds and phytoplasmas. Microbiology 147: 571–580. papaya in Cuba. Int. J. Syst. Evol. Microbiol. 55: 2451–2463. Cai, H., W. Wei, R.E. Davis, H. Chen and Y. Zhao. 2008. Genetic diversity Arocha, Y., O. Antesana, E. Montellano, P. Franco, G. Plata and P. Jones. among phytoplasmas infecting Opuntia species: virtual RFLP analysis 2007. ‘Candidatus Phytoplasma lycopersici’, a phytoplasma associated identifies new subgroups in the peanut witches’-broom phytoplasma with ‘hoja de perejil’ disease in Bolivia. Int. J. Syst. Evol. Microbiol. group. Int. J. Syst. Evol. Microbiol. 58: 1448–1457. 57: 1704–1710. Carginale, V., G. Maria, C. Capasso, E. Ionata, F. La Cara, M. Pastore, Bai, X., J. Zhang, I.R. Holford and S.A. Hogenhout. 2004. Compara- A. Bertaccini and A. Capasso. 2004. Identification of genes expressed tive genomics identifies genes shared by distantly related insect- in response to phytoplasma infection in leaves of Prunus armeniaca by transmitted plant pathogenic mollicutes. FEMS Microbiol. Lett. 235: messenger RNA differential display. Gene 332: 29–34. 249–258. Carraro, L., N. Loi and P. Ermacora. 2001. Transmission characteristics Bai, X., J. Zhang, A. Ewing, S.A. Miller, A. Jancso Radek, D.V. of the European stone fruit yellows phytoplasma and its vector Cacop- Shevchenko, K. Tsukerman, T. Walunas, A. Lapidus, J.W. Campbell sylla pruni. Eur. J. Plant Pathol. 107: 695–700. and S.A. Hogenhout. 2006. Living with genome instability: the adap- Chang, F.L., C.C. Chen and C.P. Lin. 1995. Monoclonal antibody for the tation of phytoplasmas to diverse environments of their insect and detection and identification of a phytoplasma associated with rice plant hosts. J. Bacteriol. 188: 3682–3696. yellow dwarf. Eur. J. Plant Pathol. 101: 511–518. Barbara, D.J., A. Morton, M.F. Clark and D.L. Davies. 2002. Immu- Chen, M.H. and C. Hiruki. 1978. The preservation of membranes of nodominant membrane proteins from two phytoplasmas in the tubular bodies associated with mycoplasma-like organisms by tannic aster yellows clade (chlorante aster yellows and clover phyllody) are acid. Can. J. Bot. 56: 2878–2882. Genus I. “Candidatus Phytoplasma” 713

Chen, T.A., D.A. Lei and C.P. Lin. 1989. Detection and identification Davis, R.E. and W.A. Sinclair. 1998. Phytoplasma identity and disease of plant and insect mollicutes. In The Mycoplasmas, vol. 5 (edited by etiology. Phytopathology 88: 1372–1376. Whitcomb and Tully). Academic Press, New York, pp. 393–424. Davis, R.E., R. Jomantiene, A. Kalvelyte and E.L. Dally. 2003a. Differen- Chi, K.L. and C.P. Lin. 2005. Cloning and analysis of polC gene of phy- tial amplification of sequence heterogeneous ribosomal RNA genes toplasma associated with peanut witches’ broom. Plant Pathol. Bull. and classification of the ‘Fragaria multicipita’ phytoplasma. Microbiol. 14: 51–58. Res. 158: 229–236. Chiykowski, L.N. 1983. Frozen leafhoppers as a vehicle for long-term Davis, R.E., R. Jomantiene, Y. Zhao and E.L. Dally. 2003b. Folate biosyn- storage of different isolates of the aster yellows agents. Can. J. Plant thesis pseudogenes, PsifolP and PsifolK, and an O-sialoglycoprotein Pathol. 5: 101–106. endopeptidase gene homolog in the phytoplasma genome. DNA Chiykowski, L.N. 1988. Maintenance of yellows-type mycoplasmalike Cell Biol. 22: 697–706. organisms. In Tree Mycoplasmas and Mycoplasma Diseases (edited Davis, R.E., R. Jomantiene and Y. Zhao. 2005. Lineage-specific decay of by Hiruki). The University of Alberta Press, Edmonton, Alberta, folate biosynthesis genes suggests ongoing host adaptation in phyto- ­Canada, pp. 123–134. plasmas. DNA Cell Biol. 24: 832–840. Chiykowski, L.N. and R.C. Sinha. 1990. Differentiation of MLO diseases Davis, R.E., R. Jomantiene, E. L. Dally and T. K. Wolf. 1998. Phytoplas- by means of symptomatology and vector transmission. Rec. Adv. mas associated with grapevine yellows in Virginia belong to group Mycoplasmol. Suppl. 20: 280–287. 16SrI, subgroup A (tomato big bud phytoplasma subgroup), and Christensen, N.M., M. Nicolaisen, M. Hansen and A. Schulz. 2004. Dis- group 16SrIII, new subgroup I. Vitis 37: 131–137. tribution of phytoplasmas in infected plants as revealed by real-time Denes, A.S. and R.C. Sinha. 1991. Extrachromosomal DNA elements of PCR and bioimaging. Mol. Plant Microbe. Interact. 17: 1175–1184. plant-pathogenic mycoplasma-like organisms. Can. J. Plant Pathol. Chu, Y.R., W. Y. Chen and C.P. Lin. 2006. Cloning and sequence analses 13: 26–32. of recA gene of phytoplasma associated with peanut witches’ broom. Denes, A.S. and R.C. Sinha. 1992. Alteration of clover phyllody myco- Plant Pathol. Bull. 15: 211–218. plasma DNA after in vitro culturing of phyllody-diseased clover. Can. J. Chuang, J.G. and C.P. Lin. 2000. Cloning of gyrB and gyrA genes of phy- Plant Pathol. 14: 189–196. toplasma associated with peanut witches’ broom. Plant Pathol. Bull. Deng, S. and C. Hiruki. 1991. Amplification of 16S rRNA genes from 9: 157–166. culturable and non-culturable mollicutes. J. Microbiol. Meth. 14: Cimerman, A., G. Arnaud and X. Foissac. 2006. Stolbur phytoplasma 53–61. genome survey achieved using a suppression subtractive hybridiza- Doi, Y., M. Teranaka, K. Yora and H. Asuyama. 1967. Mycoplasma or tion approach with high specificity. Appl. Environ. Microbiol. 72: PLT-group-like organisms found in the phloem elements of plants 3274–3283. infected with mulberry dwarf, potato witches-broom, aster yellows, Cimerman, A., D. Pacifico, P. Salar, C. Marzachi and X. Foissac. 2009. or Paulownia witches broom. Ann. Phytopathol. Soc. Jpn. 33: 256– Striking diversity of vmp1, a variable gene encoding a putative mem- 266. brane protein of the stolbur phytoplasma. Appl. Environ. Microbiol. Errampelli, D. and J. Fletcher. 1993. Production of monospecific poly- 75: 2951–2957. clonal antibodies made against aster yellows MLO-associated anti- Clark, M.F. 1992. Immunodiagnostic techniques for plant mycoplasma- gen. Phytopathology 83: 1279–1282. like organisms. In Techniques for the Rapid Detection of Plant Esau, K., A.C. Magyarosy and V. Breazeale. 1976. Studies of the myco- Pathogens (edited by Duncan and Torrance). Blackwell Scientific plasma-like organism (MLO) in spinach leaves affected by the aster Publications, Oxford, pp. 34–45. yellows disease. Protoplasma 90: 189–203. Cordova, I., P. Jones, N.A. Harrison and C. Oropeza. 2003. In situ PCR Evert, R.F. 1977. Phloem structure and histochemistry. Annu. Rev. Plant detection of phytoplasma DNA in embryos from coconut palms with Physiol. 28: 199–222. lethal yellowing disease. Mol. Plant Pathol. 4: 99–108. Firrao, G., C.D. Smart and B.C. Kirkpatrick. 1996. Physical map of Cousin, M.T., J. Roux, E. Boudon-Padieu, R. Berges, E. Seemüller and the western X-disease phytoplasma chromosome. J. Bacteriol. 178: C. Hiruki. 1998. Use of heteroduplex mobility analysis (HMA) for dif- 3985–3988. ferentiating phytoplasma isolates causing witches’ broom disease of Firrao, G., K. Gibb and C. Streten. 2005. Short taxonomic guide to the Populus nigra vc Italica and stolbur or big bud symptoms on tomato. genus ‘Candidatus Phytoplasma’. J. Plant Pathol. 87: 249–263. J. Phytopathol. 146: 97–102. Florance, E.R. and H.T. Cameron, 1978. Three-dimensional structure D’Arcy, C.J. and L.R. Nault. 1982. Insect transmission of plant viruses and morphology of mycoplasma-like bodies associated with albino and mycoplasmalike and rickettsialike organisms. Plant Dis. 66: disease of Prunus avium. Phytopathology 68: 75–80. 99–104. Flores, H.E., J. M. Vivanco and V.M. Loyola-Vargas. 1999. ‘Radicle’ bio- Daire, X., D. Clair, J. Larrue, E. Boudon-Padieu and A. Caudwell. 1993. chemistry: the biology of root-specific metabolism. Trends Plant Sci. Diversity among mycoplasma-like organisms inducing grapevine yel- 4: 220–226. lows in France. Vitis 32: 159–163. Galetto, L., J. Fletcher, D. Bosco, M. Turina, A. Wayadande and Davis, M.J., J.H. Tsai, R.L. Cox, L.L. McDaniel and N.A. Harrison. 1988. C. Marzachi. 2008. Characterization of putative membrane protein Cloning of chromosomal and extrachromosomal DNA of the myco- genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yel- plasma-like organism that causes maize bushy stunt disease. Mol. lows isolate. Can. J. Microbiol. 54: 341–351. Plant Microbe Interact. 1: 295–302. Garcia-Chapa, M., A. Batlle, D. Rekab, M.R. Rosquete and G. Firrao. Davis, R.E. and R.F. Whitcomb. 1970. Evidence on possible mycoplasma 2004. PCR-mediated whole genome amplification of phytoplasmas. etiology of aster yellows disease. I. Suppression of symptom develop- J. Microbiol. Methods 56: 231–242. ment in plants by antibiotics. Infect. Immun. 2: 201–208. Garnier, M., X. Foissac, P. Gaurivaud, F. Laigret, J. Renaudin, C. Saillard Davis, R.E. and I.-M. Lee. 1992. Mycoplasmalike organisms as plant dis- and J.M. Bové. 2001. Mycoplasmas, plants, insect vectors: a matrimo- ease agents. ATCC Quart. Newsl. 4: 8–11. nial triangle. C. R. Acad. Sci. III 324: 923–928. Davis, R.E. and I.-M. Lee. 1993. Cluster-specific polymerase chain reac- Gibb, K.S., B. Schneider and A.C. Padovan. 1998. Differential detection tion amplification of 16S rDNA sequences for detection and identifi- and genetic relatedness of phytoplasmas in papaya. Plant Pathol. 47: cation of mycoplasmalike organisms. Phytopathology 63: 1008–1011. 325–332. Davis, R.E., E.L. Dally, D.E. Gundersen, I.M. Lee and N. Habili. 1997. Gomez, G.G., L.R. Conci, D.A. Ducasse and S.F. Nome. 1996. Purifica- “Candidatus Phytoplasma australiense,” a new phytoplasma taxon tion of the phytoplasma associated with China-tree (Melia azedarach L.) associated with Australian grapevine yellows. Int. J. Syst. Bacteriol. decline and the production of a polyclonal antoserum for its detec- 47: 262–269. tion. J. Phytopathol. 144: 473–477. 714 Family II. Incertae sedis

Griffiths, H.M., W.A. Sinclair, C.D. Smart and R.E. Davis. 1999. The phy- ICSB Subcommittee on the Taxonomy of Mollicutes. 1997. Minutes of toplasma associated with ash yellows and lilac witches’-broom: ‘Candi- the interim meetings, 12 and 18 August, 1996, Orlando, Florida, USA datus Phytoplasma fraxini’. Int. J. Syst. Bacteriol. 49: 1605–1614. Int. J. Syst. Bacteriol. 47: 911–914. Gundersen, D.E., I.M. Lee, S.A. Rehner, R.E. Davis and D.T. Kingsbury. ICSB Subcommittee on the Taxonomy of Mollicutes. 2001. Minutes of 1994. Phylogeny of mycoplasmalike organisms (phytoplasmas): a the interim meetings, 13 and 19 July 2000, Fukuoka, Japan. Int. J. basis for their classification. J. Bacteriol. 176: 5244–5254. Syst. Evol. Microbiol. 51: 2227–2230. Gundersen, D.E. and I.M. Lee. 1996. Ultrasensitive detection of phyto- IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxon- plasmas by nested-PCR assays using two universal primer pairs. Phy- omy Group. 2004. Description of the genus ‘Candidatus Phytoplasma’, topathol. Mediterr. 35: 144–151. a taxon for the wall-less non-helical prokaryotes that colonize plant Guo, Y.H., Z.M. Cheng, J.A. Walla and Z. Zhang. 1998. Diagnosis of phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255. X-disease phytoplasma in stone fruits by a monoclonal antibody devel- Ishii, T., Y. Doi, K. Yora and H. Asuyama. 1967. Suppressive effects oped directly from a woody plant. J. Environ. Hortic. 16: 33–37. of antibiotics of tetracycline group on symptom development Guthrie, J.N., K.B. Walsh, P.T. Scott and T.S. Rasmussen. 2001. The phy- of mulberry dwarf disease. Ann. Phytopathol. Soc. Jpn. 33: 267–275. topathology of Australian papaya dieback: a proposed role for the Jagoueix-Eveillard, S., F. Tarendeau, K. Guolter, J.L. Danet, J.M. phytoplasma. Physiol. Mol. Plant Pathol. 58: 23–30. ­Bové and M. Garnier. 2001. Catharanthus roseus genes regulated Haggis, G.H. and R.C. Sinha. 1978. Scanning electron microscopy of ­differentially by mollicute infections. Mol. Plant Microbe Interact. mycoplasmalike organisms after freeze fracture of plant tissues affected 14: 225–233. with clover phyllody and aster yellows. Phytopathology 68: 677–680. Jarausch, W., C. Saillard, F. Dosba and J.M. Bové. 1994. Differentiation Hanboonsong, Y., C. Choosai, S. Panyim and S. Damak. 2002. Transo- of mycoplasmalike organisms (MLOs) in European fruit trees by PCR varial transmission of sugarcane white leaf phytoplasma in the insect using specific primers derived from the sequence of a chromosomal vector Matsumuratettix hiroglyphicus (Matsumura). Insect. Mol. Biol. fragment of the apple proliferation MLO. Appl. Environ. Microbiol. 11: 97–103. 60: 2916–2923. Harrison, N.A., J.H. Tsai, C.M. Bourne and P.A. Richardson. 1991. Jarausch, W., C. Saillard and F. Dosba. 1996. Long-term maintenance Molecular cloning and detection of chromosomal and extrachromo- of nonculturable apple proliferation phytoplasmas in their micro- somal DNA of mycoplasma-like organisms associated with witches’ propagated natural host plant. Plant Pathol. 45: 778–786. broom disease of pigeon pea in Florida. Mol. Plant Microbe Interact. Jiang, Y.P. and T.A. Chen. 1987. Purification of mycoplasma-like organisms 4: 300–307. from lettuce with aster yellows disease. Phytopathology 77: 949–953. Harrison, N.A., C.M. Bourne, R.L. Cox, J.H. Tsai and P.A. Richard- Jiang, Y.P., J.D. Lei and T.A. Chen. 1988. Purification of aster yellows son. 1992. DNA probes for detection of mycoplasma-like organisms agent from diseased lettuce using affinity chromatography. Phytopa- ­associated with lethal yellowing disease of palms in Florida. Phyto­ thology 78: 828–831. pathology 82: 216–224. Jiang, Y.P., T.A. Chen, L.N. Chiykowski and R.C. Sinha. 1989. Produc- Harrison, N.A., W. Myrie, P. Jones, M.L. Carpio, M. Castillo, M.M. Doyle tion of monoclonal antibodies to peach eastern-X disease and their and C. Oropeza. 2002. 16S rRNA interoperon sequence heterogene- use in disease detection. Can. J. Plant Pathol. 11: 325–331. ity distinguishes strain populations of palm lethal yellowing phyto- Jomantiene, R., R.E. Davis, D. Valiunas and A. Alminaite. 2002. New group plasma in the Caribbean region. Ann. Appl. Biol. 141: 183–193. 16SrIII phytoplasma lineages in Lithuania exhibit rRNA interoperon Harrison, N.A., E. Boa and M.L. Carpio. 2003. Characterization of phy- sequence heterogeneity. Eur. J. Plant Pathol. 108: 507–517. toplasmas detected in Chinaberry trees with symptoms of leaf yellow- Jomantiene, R. and R.E. Davis. 2006. Clusters of diverse genes existing ing and decline in Bolivia. Plant Pathol. 52: 147–157. as multiple, sequence-variable mosaics in a phytoplasma genome. Hearon, S.S., R.H. Lawson, F.F. Smith, J.T. Mckenzie and J. Rosen. 1976. FEMS Microbiol. Lett. 255: 59–65. Morphology of filamentous forms of a mycoplasmalike organism Jomantiene, R., Y. Zhao and R.E. Davis. 2007. Sequence-variable mosaics: associated with hydrangea virescence. Phytopathology 66: 608–616. composites of recurrent transposition characterizing the genomes of Hiruki, C. and K. Wang. 2004. Clover proliferation phytoplasma: phylogenetically diverse phytoplasmas. DNA Cell Biol. 26: 557–564. ‘Candidatus Phytoplasma trifolii’. Int. J. Syst. Evol. Microbiol. 54: Jones, P. 2002. Phytoplasma plant pathogens. In Plant Pathologists 1349–1353. Pocketbook (edited by Waller). CAB International, Wallingford, UK, Ho, K.C., C.C. Tsai and T.L. Chung. 2001. Organization of ribosomal pp. 126–139. RNA genes from a Loofah witches’ broom phytoplasma. DNA Cell Jung, H.Y., T. Sawayanagi, S. Kakizawa, H. Nishigawa, S. Miyata, Biol. 20: 115–122. K. Oshima, M. Ugaki, J.T. Lee, T. Hibi and S. Namba. 2002. ‘Candi- Hodgetts, J., N. Boonham, R. Mumford, N. Harrison and M. Dickin- datus Phytoplasma castaneae’, a novel phytoplasma taxon associated son. 2008. Phytoplasma phylogenetics based on analysis of secA and with chestnut witches’ broom disease. Int. J. Syst. Evol. Microbiol. 52: 23S rRNA gene sequences for improved resolution of candidate 1543–1549. species of ‘Candidatus Phytoplasma’. Int. J. Syst. Evol. Microbiol. 58: Jung, H.Y., S. Miyata, K. Oshima, S. Kakizawa, H. Nishigawa, W. Wei, S. 1826–1837. Suzuki, M. Ugaki, T. Hibi and S. Namba. 2003a. First complete nucle- Hogenhout, S.A., K. Oshima, D. Ammar el, S. Kakizawa, H.N. Kingdom otide sequence and heterologous gene organization of the two rRNA and S. Namba. 2008. Phytoplasmas: bacteria that manipulate plants operons in the phytoplasma genome. DNA Cell Biol. 22: 209–215. and insects. Mol. Plant. Pathol. 9: 403–423. Jung, H.Y., T. Sawayanagi, S. Kakizawa, H. Nishigawa, W. Wei, Hren, M., M. Ravnikar, J. Brzin, P. Ermacora, L. Carraro, P. A. Bianco, K. Oshima, S. Miyata, M. Ugaki, T. Hibi and S. Namba. 2003b. ‘Can- P. Casati, M. Borgo, E. Angelini, A. Rotter and K. Gruden. 2009. didatus Phytoplasma ziziphi’, a novel phytoplasma taxon associated Induced expression of sucrose synthase and alcohol dehydrogenase with jujube witches’-broom disease. Int. J. Syst. Evol. Microbiol. 53: I genes in phytoplasma-infected grapevine plants grown in the field. 1037–1041. Plant Pathol. 58: 170–180. Jung, H.Y., T. Sawayanagi, P. Wongkaew, S. Kakizawa, H. Nishigawa, Hsu, H.T., I.M. Lee, R.E. Davis and Y.C. Wang. 1990. Immunization for W. Wei, K. Oshima, S. Miyata, M. Ugaki, T. Hibi and S. Namba. 2003c. generation of hybridoma antibodies specifically reacting with plants ‘Candidatus Phytoplasma oryzae’, a novel phytoplasma taxon associated infected with a mycoplasmalike organism (MLO) and their use in with rice yellow dwarf disease. Int. J. Syst. Evol. Microbiol. 53: 1925–1929. detection of MLO antigens. Phytopathology 80: 946–950. Kakizawa, S., K. Oshima, T. Kuboyama, H. Nishigawa, H. Jung, ICSB Subcommittee on the Taxonomy of Mollicutes. 1993. Minutes of T. Sawayanagi, T. Tsuchizaki, S. Miyata, M. Ugaki and S. Namba. 2001. the Interim meetings, 1 and 2 August, 1992, Ames, Iowa. Int. J. Syst. Cloning and expression analysis of Phytoplasma protein translocation Bacteriol. 43: 394–397. genes. Mol. Plant Microbe Interact. 14: 1043–1050. Genus I. “Candidatus Phytoplasma” 715

Kakizawa, S., K. Oshima, H. Nighigawa, H.Y. Jung, W. Wei, S. Suzuki, Kunkel, L.O. 1926. Studies on aster yellows. Am. J. Bot. 13: 646–705. M. Tanaka, S. Miyata, M. Ugaki and S. Namba. 2004. Secretion of Kuske, C.R. and B.C. Kirkpatrick. 1990. Identification and character- immunodominant membrane protein from onion yellows phyto- ization of plasmids from the western aster yellows mycoplasmalike plasma through the Sec protein-translocation system in Escherichia organism. J. Bacteriol. 172: 1628–1633. coli. Microbiology 150: 135–142. Kuske, C.R., B.C. Kirkpatrick and E. Seemüller. 1991. Differentiation of Kakizawa, S., K. Oshima, Y. Ishii, A. Hoshi, K. Maejima, H.Y. Jung, Y. virescence MLOS using western aster yellows mycoplasma-like organ- Yamaji and S. Namba. 2009. Cloning of immunodominant mem- ism chromosomal DNA probes and restriction fragment length poly- brane protein genes of phytoplasmas and their in planta expression. morphism analysis. J. Gen. Microbiol. 137: 153–159. FEMS Microbiol. Lett. 293: 92–101. Kuske, C.R. and B.C. Kirkpatrick. 1992a. Distribution and multiplication Kamin´ska, M. and H. liwa. 2003. Effect of antibiotics on symptoms of western aster yellows mycoplasmalike organisms in Catharanthus of stunting disease of Magnolia lilliflora plants. J. Phytopathol. 151: roseus as determined by DNA hybridization analysis. Phytopathology 59–63. 82: 457–462. Kawakita, H., T. Saiki, W. Wei, W. Mitsuhashi, K. Watanabe and M. Sato. Kuske, C.R. and B.C. Kirkpatrick. 1992b. Phylogenetic relationships 2000. Identification of mulberry dwarf phytoplasmas in the genital between the western aster yellows mycoplasmalike organism and organs and eggs of leafhopper Hishimonoides sellatiformis. Phytopa- other prokaryotes established by 16S rRNA gene sequence. Int. J. thology 90: 909–914. Syst. Bacteriol. 42: 226–233. Kenyon, L., N.A. Harrison, G.R. Ashburner, E.R. Boa and P.A. Richard- Lauer, U. and E. Seemüller. 2000. Physical map of the chromosome of son. 1998. Detection of a pigeon pea witches’ broom-related phyto- the apple proliferation phytoplasma. J. Bacteriol. 182: 1415–1418. plasma in trees of Gliricidia sepium affected by little-leaf disease in Lee, I.-M. and R.E. Davis. 1992. Mycoplasmas which infect plants and Central America. Plant Pathol. 47: 671–680. insects. In Mycoplasmas: Molecular Biology and Pathogenesis (edited Khan, A.J., S. Botti, S. Paltrinieri, A.M. Al-Subhi and A.F. Bertaccina. 2002. by Maniloff, McElhaney, Finch and Baseman). American Society for Phytoplasmas in alfalfa seedlings: infected or contaminated seed? Microbiology, Washington, D.C., pp. 379–390. Proceedings of the 14th International Congress of the ­International Lee, I.-M., A. Bertaccini, M. Vibio and D.E. Gundersen. 1988. Detec- Organization for Mycoplasmology, Vienna, Austria, p. 148. tion and investigation of genetic relatedness among aster yellows Kirkpatrick, B.C., D.C. Stenger, T.J. Morris and A.H. Purcell. 1987. and other mycoplasmalike organisms by using cloned DNA and RNA Cloning and detection of DNA from a nonculturable plant patho- probes. Mol. Plant Microbe Interact. 1: 303–310. genic Mycoplasma-like organism. Science 238: 197–200. Lee, I.-M., R.E. Davis, T.A. Chen, L.N. Chiykowski, J. Fletcher, C. Hiruki Kirkpatrick, B.C. 1989. Strategies for characterizing plant pathogenic and D.A. Schaff. 1992. A genotype-based system for identification mycoplasma-like organisms and their effects on plants. In Plant- and classification of mycoplasmalike organisms (MLOs) in the aster Microbe Interactions, Molecular and Genetic Perspectives (edited by yellows MLO strain cluster. Phytopathology 82: 977–986. Kosuge and Nester). McGraw-Hill, New York, pp. 241–293. Lee, I.-M., R.E. Davis and H.T. Hsu. 1993a. Differentiation of strains in Kirkpatrick, B.C. 1992. Mycoplasma-like organisms: plant and inverte- the aster yellows mycoplasmalike organism strain cluster by serologi- brate pathogens. In The Prokaryotes: a Handbook on the Biology of cal assay with monoclonal antibodies. Plant Dis. 77: 815–817. Bacteria: Ecophysiology, Isolation, Identification, Applications, 2nd Lee, I.-M., R.W. Hammond, R.E. Davis and D.E. Gundersen. 1993b. edn, vol. 4 (edited by Balows, Trüper, Dworkin, Harder and Schle- Universal amplification and analysis of pathogen 16S rDNA for clas- ifer). Springer, New York, pp. 4050–4067. sification and identification of mycoplasmalike organisms. Phytopa- Kirkpatrick, B.C., N.A. Harrison, I.-M. Lee, H. Neimark and B.B. Sears. thology 83: 834–842. 1995. Isolation of Mycoplasma-like organism DNA from plant and Lee, I.-M., A. Bertaccini, M. Vibio and D.E. Gundersen. 1995. Detection insect hosts. In Molecular and Diagnostic Procedures in Mycoplas- of multiple phytoplasmas in perennial fruit trees with decline symp- mology, vol. 2 (edited by Razin and Tully). Academic Press, New toms in Italy. Phytopathology 85: 728–735. York, pp. 105–117. Lee, I.-M., D.E. Gundersen-Rindal and A. Bertaccini. 1998a. ­Phytoplasma: Kison, H., B. Schneider and E. Seemüller. 1994. Restriction fragment ecology and genomic diversity. Phytopathology 88: 1359–1366. length polymorphisms within the apple proliferation mycoplasma- Lee, I.-M., D.E. Gundersen-Rindal, R.E. Davis and I.M. Bartoszyk. like organism. J. Phytopathol. 141: 395–401. 1998b. Revised classification scheme of phytoplasmas based an RFLP Kison, H., B.C. Kirkpatrick and E. Seemüller. 1997. Genetic comparison analyses of 16S rRNA and ribosomal protein gene sequences. Int. J. of the peach yellow leaf roll agent with European fruit tree phytoplas- Syst. Bacteriol. 48: 1153–1169. mas of the apple proliferation group. Plant Pathol. Bull. 46: 538–544. Lee, I.-M., R.E. Davis and D.E. Gundersen-Rindal. 2000. Phytoplasma: Kollar, A. and E. Seemüller. 1989. Base composition of the DNA of phytopathogenic Mollicutes. Annu. Rev. Microbiol. 54: 221–255. mycoplasmalike organisms associated with various plant diseases. Lee, I.-M., M. Martini, K.D. Bottner, R.A. Dane, M.C. Black and N. Trox- J. Phytopathol. 127: 177–186. clair. 2003. Ecological implications from a molecular analysis of phy- Kollar, A. and E. Seemüller. 1990. Chemical composition of the phloem toplasmas involved in an aster yellows epidemic in various crops in exudate of Mycoplasma-infected trees. J. Phytopathol. 128: 99–111. Texas. Phytopathology 93: 1368–1377. Koui, T., T. Natsuaki and S. Okuda. 2002. Antiserum raised against Lee, I.-M., D.E. Gundersen-Rindal, R.E. Davis, K.D. Bottner, C. Marcone gyrase A of Acholeplasma laidlawii reacts with phytoplasma proteins. and E. Seemüller. 2004a. ‘Candidatus Phytoplasma asteris’, a novel FEMS Microbiol. Lett. 206: 169–174. phytoplasma taxon associated with aster yellows and related diseases. Koui, T., N. Tomohide and S. Okuda. 2003. Phylogenetic analysis of Int. J. Syst. Evol. Microbiol. 54: 1037–1048. elongation factor Tu gene of phytoplasmas from Japan. J. Gen. Plant Lee, I.-M., M. Martini, C. Marcone and S.F. Zhu. 2004b. Classification of Pathol. 69: 316–319. phytoplasma strains in the elm yellows group (16SrV) and proposal Kube, M., B. Schneider, H. Kuhl, T. Dandekar, K. Heitmann, A. M. Mig- of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated doll, R. Reinhardt and E. Seemüller. 2008. The linear chromosome with elm yellows. Int. J. Syst. Evol. Microbiol. 54: 337–347. of the plant-pathogenic Mycoplasma ‘Candidatus Phytoplasma mali’. Lee, I.-M., Y. Zhao and K.D. Bottner. 2005. Novel insertion sequence-like BMC Genomics 9: 306. elements in phytoplasma strains of the aster yellows group are putative Kuboyama, T., C.C. Huang, X. Lu, T. Sawayanagi, T. Kanazawa, new members of the IS3 family. FEMS Microbiol. Lett. 242: 353–360. T. Kagami, I. Matsuda, T. Tsuchizaki and S. Namba. 1998. A plas- Lee, I.-M., K.D. Bottner, G. Secor and V. Rivera-Varas. 2006a. “Candi- mid isolated from phytopathogenic onion yellows phytoplasma and datus Phytoplasma americanum”, a phytoplasma associated with a its heterogeneity in the pathogenic phytoplasma mutant. Mol. Plant potato purple top wilt disease complex. Int. J. Syst. Evol. Microbiol. Microbe Interact. 11: 1031–1037. 56: 1593–1597. 716 Family II. Incertae sedis

Lee, I.-M., Y. Zhao and K.D. Bottner. 2006b. SecY gene sequence analy- Marcone, C. and E. Seemüller. 2001. A chromosome map of the sis for finer differentiation of diverse strains in the aster yellows phy- European stone fruit yellows phytoplasma. Microbiology 147: toplasma group. Mol. Cell. Probes 20: 87–91. 1213–1221. Lefol, C., J. Lherminier, E. Boudon-Padieu, J. Larrue, C. Louis and A. Marcone, C., K.S. Gibb, C. Streten and B. Schneider. 2004a. ‘Candidatus Caudwell. 1994. Propagation of Flavescence Dorèe MLO (myco- Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candi- plasma-like organisms) in the leafhopper vector Euscelidius variega- datus Phytoplasma allocasuarinae’, respectively associated with spar- tus. Kbm. J. Invertebr. Pathol. 63: 285–293. tium witches’-broom, buckthorn witches’-broom and allocasuarina Lepka, P., M. Stitt, E. Moll and E. Seemüller. 1999. Effect of phytoplas- yellows diseases. Int. J. Syst. Evol. Microbiol. 54: 1025–1029. mal infection on concentration and translocation of carbohydrates Marcone, C., B. Schneider and E. Seemüller. 2004b. ‘Candidatus Phyto- and amino acids in periwinkle and tobacco. Physiol. Mol. Plant plasma cynodontis’, the phytoplasma associated with Bermuda grass Pathol. 55: 59–68. white leaf disease. Int. J. Syst. Evol. Microbiol. 54: 1077–1082. Liefting, L.W., M.T. Andersen, R.E. Beever, R.C. Gardner and R.L.S. Martinez, S., I. Cordova, B.E. Maust, C. Oropeza and J.M. Santamaria. Forster. 1996. Sequence heterogeneity in the two 16S rRNA genes 2000. Is abscisic acid responsible for abnormal stomatal closure in coco- of Phormium yellow leaf phytoplasma. Appl. Environ. Microbiol. 62: nut palms showing lethal yellowing? J. Plant Physiol. 156: 319–322. 3133–3139. Martini, M., I.M. Lee, K.D. Bottner, Y. Zhao, S. Botti, A. Bertaccini, Liefting, L.W. and B.C. Kirkpatrick. 2003. Cosmid cloning and sample N.A. Harrison, L. Carraro, C. Marcone, A.J. Khan and R. Osler. 2007. sequencing of the genome of the uncultivable mollicute, western Ribosomal protein gene-based phylogeny for finer differentiation X-disease phytoplasma, using DNA purified by pulsed-field gel elec- and classification of phytoplasmas. Int. J. Syst. Evol. Microbiol. 57: trophoresis. FEMS Microbiol. Lett. 221: 203–211. 2037–2051. Liefting, L.W., M.E. Shaw and B.C. Kirkpatrick. 2004. Sequence analysis Marzachi, C., R.G. Milne and D. Bosco. 2004. Phytoplasma-plant- of two plasmids from the phytoplasma beet leafhopper-transmitted vector relationships. In Recent Research Developments in Plant virescence agent. Microbiology 150: 1809–1817. Pathology, vol. 3 (edited by Pandalai). Research Signpost, Trivan- Liefting, L.W., M.T. Andersen, T.J. Lough and R.E. Beever. 2006. Com- drum, India. parative analysis of the plasmids from two isolates of “Candidatus Phy- McCoy, R.E. 1979. Mycoplasmas and yellows diseases. In The Mycoplas- toplasma australiense”. Plasmid 56: 138–144. mas, vol. III, Plant and Insect Mycoplasmas (edited by Whitcomb and Lim, P.O. and B.B. Sears. 1989. 16S rRNA sequence indicates that plant- Tully). Academic Press, New York, pp. 229–264. pathogenic mycoplasmalike organisms are evolutionarily distinct McCoy, R.E. 1982. Use of tetracycline antibiotics to control yellows dis- from animal mycoplasmas. J. Bacteriol. 171: 5901–5906. eases. Plant Dis. 66: 539–542. Lim, P.O. and B.B. Sears. 1992. Evolutionary relationships of a plant- McCoy, R.E., A. Caudwell, C.J. Chang, T.A. Chen, L.N. Chiykowski, pathogenic mycoplasmalike organism and Acholeplasma laidlawii M.T. Cousin, J.L. Dale, G.T.N. deLeeuw, D.A. Golino, K.J. Hackett, deduced from two ribosomal protein gene sequences. J. Bacteriol. B.C. Kirkpatrick, R. Marwitz, H. Petzold, R.C. Sinha, M. Suguira, 174: 2606–2611. R.F. Whitcomb, I.L. Yang, B.M. Zhu and E. Seemüller. 1989. Plant Lim, P.O., B.B. Sears and K.L. Klomparens. 1992. Membrane properties diseases associated with mycoplasma-like organisms. In The Myco- of a plant-pathogenic mycoplasmalike organism. J. Bacteriol. 174: plasmas, vol. V (edited by Whitcomb and Tully). Academic Press, San 682–686. Diego, pp. 545–640. Lin, C.-L., T. Zhou, H.-F. Li, Z.-F. Fan, Y. Li, C.-G. Piao and G.-Z. Tian. Melamed, S., E. Tanne, R. Ben-Haim, O. Edelbaum, D. Yogev and 2009. Molecular characterisation of two plasmids from paulownia I. Sela. 2003. Identification and characterization of phytoplasmal witches’-broom phytoplasma and detection of a plasmid encoded genes, employing a novel method of isolating phytoplasmal genomic protein in infected plants. Eur. J. Plant Pathol. 123: 321–330. DNA. J. Bacteriol. 185: 6513–6521. Lin, C.-Y., Chen, W.-Y., and C.P. Lin. 2006. Cloning and analysis of rpoC Miyata, S., K. Furuki, K. Oshima, T. Sawayanagi, H. Nishigawa, S. Kak- gene of phytoplasma associated with peanut witches’ broom. Plant izawa, H.Y. Jung, M. Ugaki and S. Namba. 2002a. Complete nucleotide Pathol. Bull. 15: 129–138. sequence of the S10-spc operon of phytoplasma: Gene organization Loi, N., P. Ermacora, T.A. Chen, L. Carraro and R. Osler. 1998. Mono- and genetic code resemble those of Bacillus subtilis. DNA Cell Biol. clonal antibodies for the detection of tagetes witches’ broom agent. 21: 527–534. J. Plant Pathol. 80: 171–174. Miyata, S., K. Furuki, T. Sawayanagi, K. Oshima, T. Kuboyama, Loi, N., P. Ermacora, L. Carraro, R. Osler and T.A. Chen. 2002. Produc- T. Tsuchizaki, M. Ugaki and S. Namba. 2002b. The gene arrangement tion of monoclonal antibodies against apple proliferation phytoplasma and sequence of str operon of phytoplasma resemble those of Bacillus and their use in serological detection. Eur. J. Plant Pathol. 108: 81–86. more than those of Mycoplasma. J. Gen. Plant Pathol. 68: 62–67. Marcone, C., A. Ragozzino, B. Schneider, U. Lauer, C.D. Smart and Miyata, S., K. Oshima, S. Kakizawa, H. Nishigawa, H.Y. Jung, E. Seemüller. 1996. Genetic characterization and classification of two T. Kuboyama, M. Ugaki and S. Namba. 2003. Two different thymidy- phytoplasmas associated with spartium witches’ broom disease. Plant late kinase gene homologues, including one that has catalytic activity, Dis. 80: 365–371. are encoded in the onion yellows phytoplasma genome. Microbiol- Marcone, C., F. Hergenhahn, A. Ragozzino and E. Seemüller. 1999a. ogy 149: 2243–2250. Dodder transmission of pear decline, European stone fruit yellows, Montano, H.G., R.E. Davis, E.L. Dally, S. Hogenhout, J.P. Pimentel and rubus stunt, Picris echioides yellows and cotton phyllody phytoplasmas P.S. Brioso. 2001. ‘Candidatus Phytoplasma brasiliense’, a new phyto- to periwinkle. J. Phytopathol. 147: 187–192. plasma taxon associated with hibiscus witches’ broom disease. Int. J. Marcone, C., H. Neimark, A. Ragozzino, U. Lauer and E. Seemüller. Syst. Evol. Microbiol. 51: 1109–1118. 1999b. Chromosome sizes of phytoplasmas composing major phylo- Moriwaki, N., K. Matsuchita, M. Nishina and Y. Kono. 2003. High con- genetic groups and subgroups. Phytopathology 89: 805–810. centrations of trehalose in aphid hemolymph Appl. Entomol. Zool. Marcone, C., I.-M. Lee, R.E. Davis, A. Ragozzino and E. Seemüller. 38: 241–248. 2000. Classification of aster yellows-group phytoplasmas based on Morton, A., D.L. Davies, C.L. Blomquist and D.J. Barbara. 2003. Char- combined analyses of rRNA and tuf gene sequences. Int. J. Syst. Evol. acterization of homologues of the apple proliferation immunodomi- Microbiol. 50: 1703–1713. nant membrane protein gene from three related phytoplasmas. Mol. Marcone, C., A. Ragozzino, I. Camele, G.L. Rana and E. Seemüller. Plant Pathol. 4: 109–114. 2001. Updating and extending genetic characterization and classifi- Mounsey, K.E., C. Streten and K.S. Gibb. 2006. Sequence character- cation of phytoplasmas from wild and cultivated plants in southern ization of four putative membrane-associated proteins from sweet Italy. J. Plant Pathol. 83: 133–138. potato little strain V4 phytoplasma. Plant Pathol. 55: 29–35. Genus I. “Candidatus Phytoplasma” 717

Murray, R.G.E., D.J. Brenner, R.R. Colwell, P. de Vos, P. Goodfellow, Oshima, K., T. Shiomi, T. Kuboyama, T. Sawayanagi, H. Nishigawa, P.A.D. Grimont, N. Pfennig, E. Stackebrandt and G.A. Zavarin. 1990. S. Kakizawa, S. Miyata, M. Ugaki and S. Namba. 2001b. Isolation and Report of the ad hoc committee on approaches to taxonomy within characterization of derivative lines of the onion yellows phytoplasma the proteobacteria. Int. J. Syst. Bacteriol. 40: 213–215. that do not cause stunting or phloem hyperplasia. Phytopathology Murray, R.G.E. and K.H. Schleifer. 1994. Taxonomic notes: a proposal 91: 1024–1029. for recording the properties of putative taxa of procaryotes. Int. J. Oshima, K., S. Miyata, T. Sawayanagi, S. Kakizawa, H. Nishigawa, H. Jung, Syst. Bacteriol. 44: 174–176. K. Furuki, M. Yanazaki, S. Suzuki, W. Wei, T. Kuboyama, M. Ugaki and Musetti, R., M.A. Favali and L. Pressacco. 2000. Histopathology and S. Namba. 2002. Minimal set of metabolic pathways suggested from the polyphenol content in plants infected by phytoplasmas. Cytobios genome of onion yellow phytoplasma. J. Plant Pathol. 68: 225–236. 102: 133–147. Oshima, K., S. Kakizawa, H. Nishigawa, H.Y. Jung, W. Wei, S. Suzuki, Musetti, R. and M.A. Favali. 2003. Cytochemical localization of calcium R. Arashida, D. Nakata, S. Miyata, M. Ugaki and S. Namba. 2004. and X-ray microanalysis of Catharanthus roseus L. infected with phyto- Reductive evolution suggested from the complete genome sequence plasmas. Micron 34: 387–393. of a plant-pathogenic phytoplasma. Nat. Genet. 36: 27–29. Musetti, R., L.S. Di Toppi, P. Ermacora and M.A. Favali. 2004. Recov- Oshima, K., S. Kakizawa, R. Arashida, Y. Ishii, A. Hoshi, Y. Hayashi, ery in apple trees infected with the apple proliferation phyto- S. Kakiwada and S. Namba. 2007. Presence of two glycolytic gene plasma: an ultrastructural and biochemical study. Phytopathology clusters in a severe pathogenic line of Candidatus Phytoplasma ­asteris. 94: 203–208. Mol. Plant Pathol. 8: 481–489. Nakashima, K. and T. Hayashi. 1997. Sequence analysis of extrachromo- Padovan, A.C., G. Firrao, B. Schneider and K.S. Gibb. 2000. Chromosome somal DNA of sugarcane white leaf phytoplasma. Ann. Phytopathol. mapping of the sweet potato little leaf phytoplasma reveals genome Soc. Jpn. 63: 21–25. heterogeneity within the phytoplasmas. Microbiology 146: 893–902. Namba, S., H. Oyaizu, S. Kato, S. Iwanami and T. Tsuchizaki. 1993. Phy- Pracros, P., J. Renaudin, S. Eveillard, A. Mouras and M. Hernould. 2006. logenetic diversity of phytopathogenic mycoplasmalike organisms. Tomato flower abnormalities induced by stolbur infection are associ- Int. J. Syst. Bacteriol. 43: 461–467. ated with changes of expression of floral development genes. Mol. Namba, S. 2002. Molecular biological studies on phytoplasmas. J. Gen. Plant Microbe Interact. 19: 62–68. Plant Pathol. 68: 257–259. Rajan, J., M.F. Clark, M. Barba and A. Hadidi. 1995. Detection of apple Nasu, S., D.D. Jensen and J. Richardson. 1970. Electron microscopy proliferation and other MLOs by immuno-capture PCR (IC-PCR). of mycoplasma-like bodies associated with insect and plant hosts of Acta Hortic. 386: 511–514. peach western X-disease. Virology 41: 583–595. Raju, B.C. and G. Nyland. 1988. Chemotherapy of mycoplasma diseases Nasu, S., D. D. Jensen and J. Richardson. 1974. Primary culture of the of fruit trees. In Tree Mycoplasmas and Mycoplasma Diseases (edited western X-disease mycoplasma-like organism from Colladonus monta- by Hiruki). University of Alberta Press, Edmonton, Alberta, Canada, nus leafhopper vectors. Appl. Entomol. Zool. 9: 115–126. pp. 207–216. Neimark, H. and B.C. Kirkpatrick. 1993. Isolation and characterization Reinert, W. 1999. Detection and further differentiation of plant patho- of full-length chromosomes from non-culturable plant-pathogenic genic phytoplasmas (Mollicutes, Eubacteria) in Germany regarding mycoplasma-like organisms. Mol. Microbiol. 7: 21–28. phytopathological aspects. PhD Dissertation. Dem Fachbereich Biol- Nielson, M.W. 1979. Taxonomic relationships of leafhopper vectors of plant ogie der Technischen Universität Darmstadt (in German), p. 148. pathogens. In Leafhopper Vectors and Plant Disease Agents (edited by Rekab, D., L. Carraro, B. Schneider, E. Seemüller, J. Chen, C.J. Chang, Maromorosch and Harris). Academic Press, New York, pp. 3–27. R. Locci and G. Firrao. 1999. Geminivirus-related extrachromosomal Nipah, J.O., P. Jones and M.J. Dickinson. 2007. Detection of lethal yel- DNAs of the X-clade phytoplasmas share high sequence similarity. lowing phytoplasmas in embryos from coconuts infected with Cape Microbiology 145: 1453–1459. St. Paul wilt disease in Ghana. Plant Pathol. 56: 777–784. Rudzinska-Langwald, A. and M. Kaminska. 1999. Cytopathological evidence Nishigawa, H., S. Miyata, K. Oshima, T. Sawayanagi, A. Komoto, for transport of phytoplasma in infected plants. Bot. Pol. 68: 261–266. T. Kuboyama, I. Matsuda, T. Tsuchizaki and S. Namba. 2001. In planta Rudzinska-Langwald, A. and M. Kaminska. 2003. Changes in the ultra- expression of a protein encoded by the extrachromosomal DNA of a structure and cytoplasmic free calcium in Gladiolus x hybridus Van phytoplasma and related to geminivirus replication proteins. Micro- Houtte roots infected by aster yellows phytoplasma. Acta Soc. Bot. biology 147: 507–513. Pol. 72: 269–282. Nishigawa, H., K. Oshima, S. Kakizawa, H.Y. Jung, T. Kuboyama, Saglio, P.H.M. and R.F. Whitcomb. 1979. Diversity of wall-less prokary- S. Miyata, M. Ugaki and S. Namba. 2002a. A plasmid from a non-insect- otes in plant vascular tissue, fungi and invertebrate animals. In The transmissible line of a phytoplasma lacks two open reading frames that Mycoplasmas, vol. 3 (edited by Whitcomb and Tully). Academic exist in the plasmid from the wild-type line. Gene 298: 195–201. Press, New York, pp. 1–36. Nishigawa, H., K. Oshima, S. Kakizawa, H.Y. Jung, T. Kuboyama, S. Sawayanagi, T., N. Horikoshi, T. Kanehira, M. Shinohara, A. Bertaccini, Miyata, M. Ugaki and S. Namba. 2002b. Evidence of intermolecular M.T. Cousin, C. Hiruki and S. Namba. 1999. ‘Candidatus Phytoplasma recombination between extrachromosomal DNAs in phytoplasma: a japonicum’, a new phytoplasma taxon associated with Japanese trigger for the biological diversity of phytoplasma? Microbiology 148: Hydrangea phyllody. Int. J. Syst. Bacteriol. 49: 1275–1285. 1389–1396. Schneider, B., U. Ahrens, B.C. Kirkpatrick and E. Seemüller. 1993. Nishigawa, H., K. Oshima, S. Miyata, M. Ugaki and S. Namba. 2003. Classification of plant-pathogenic mycoplasma-like organisms using Complete set of extrachromosomal DNAs from three pathogenic restriction-site analysis of PCR-amplified 16S rDNA. J. Gen. Micro- lines of onion yellows phytoplasma and use of PCR to differentiate biol. 139: 519–527. each line. J. Gen. Plant Pathol. 69: 194–198. Schneider, B. and E. Seemüller. 1994a. Presence of two sets of ribo- Nyland, G. 1971. Remission of symptoms of pear decline in pear and somal genes in phytopathogenic mollicutes. Appl. Environ. Micro- peach X-disease in peach after treatment with a tetracycline. Phyto- biol. 60: 3409–3412. pathology 61: 904–905. Schneider, B. and E. Seemüller. 1994b. Studies on taxonomic relation- Oparka, K.J. and R. Turgeon. 1999. Sieve elements and companion ships of mycoplasma-like organisms by Southern blot analysis. J. Phy- cells-traffic control centers of the phloem. Plant Cell. 11: 739–750. topathol. 141: 173–185. Oshima, K., S. Kakizawa, H. Nishigawa, T. Kuboyama, S. Miyata, Schneider, B., E. Seemüller, C.D. Smart and B.C. Kirkpatrick. 1995. Phy- M. Ugaki and S. Namba. 2001a. A plasmid of phytoplasma encodes logenetic classification of plant pathogenic mycoplasmalike organ- a unique replication protein having both plasmid- and virus-like isms or phytoplasmas. In Molecular and Diagnostic Procedures in domains: clue to viral ancestry or result of virus/plasmid recombina- Mycoplasmology: Molecular Characterization, vol. 1 (edited by Razin tion? Virology 285: 270–277. and Tully). Academic Press, San Diego, pp. 369–380. 718 Family II. Incertae sedis

Schneider, B., K.S. Gibb and E. Seemüller. 1997. Sequence and RFLP Stackebrandt, E. and B.M. Goebel. 1994. Taxonomic note: a place for analysis of the elongation factor Tu gene used in differentiation and DNA–DNA reassociation and 16S rRNA sequence analysis in the pres- classification of phytoplasmas. Microbiology 143: 3381–3389. ent species definition in bacteriology. Int. J. Syst. Bacteriol. 44: 846–849. Schneider, B., E. Torres, M.P. Martin, M. Schroder, H.D. Behnke and Streten, C. and K.S. Gibb. 2003. Identification of genes in the tomato E. Seemuller. 2005. ‘Candidatus Phytoplasma pini’, a novel taxon big bud phytoplasma and comparison to those in sweet potato little from Pinus silvestris and Pinus halepensis. Int. J. Syst. Evol. Microbiol. leaf-V4 phytoplasma. Microbiology 149: 1797–1805. 55: 303–307. Suzuki, S., K. Oshima, S. Kakizawa, R. Arashida, H.Y. Jung, Y. Yamaji, H. Nishi- Sears, B.B. and K.L. Klomparens. 1989. Leaf tip cultures of the evening gawa, M. Ugaki and S. Namba. 2006. Interaction between the membrane primrose allow stable, aspectic culture of mycoplasma-like organism. protein of a pathogen and insect microfilament ­complex determines Can. J. Plant Pathol. 11: 343–348. insect-vector specificity. Proc. Natl. Acad. Sci. U. S. A. 103: 4252–4257. Sears, B.B. and B.C. Kirkpatrick. 1994. Unveiling the evolutionary rela- Swofford, D.L. 1998. PAUP: Phylogenetic analysis using parsimony and tionships of plant pathogenic mycoplasmalike organisms. ASM News other methods, 4th edn. Sinauer Associates, Sunderland, MA. 60: 307–312. Tan, P.K. and T. Whitlow. 2001. Physiological responses of Catharanthus Seddas, A., R. Meignoz, C. Kuszala and E. Boudon-Padieu. 1995. Evi- roseus (periwinkle) to ash yellows phytoplasmal infection. New Phy- dence for the physical integrity of flavescence dorée phytoplasmas tol. 150: 759–769. purified by affinity chromatography by immunoaffinity from infected Tanne, E., E. Boudon-Padieu, D. Clair, M. Davidovich, S. Melamed and plants or leafhoppers and the plant pathogenicity of phytoplasmas M. Klein. 2001. Detection of phytoplasma by polymerase chain reac- from leafhoppers. Plant Pathol. 44: 971–978. tion of insect feeding medium and its use in determining vectoring Seemüller, E. and B. Schneider. 2007. Differences in virulence and ability. Phytopathology 91: 741–746. genomic features of strains of ‘Candidatus Phytoplasma mali’, the Tedeschi, R., V. Ferrato, J. Rossi and A. Alma. 2006. Possible phyto- Apple Proliferation agent. Phytopathology 97: 964–970. plasma transovarial transmission in the psyllids Cacopsylla melanon- Seemüller, E., B. Schneider, R. Mäurer, U. Ahrens, X. Daire, H. Kison, K.H. eura and Cacopsylla pruni. Plant Pathol. 55: 18–24. Lorenz, G. Firrao, L. Avinent, B.B. Sears and E. Stackebrandt. 1994. Thomas, D.L. 1979. Mycoplasmalike bodies associated with lethal Phylogenetic classification of phytopathogenic mollicutes by sequence declines of palms in Florida. Phytopathology 69: 928–934. analysis of 16S ribosomal DNA. Int. J. Syst. Bacteriol. 44: 440–446. Thomas, S. and M. Balasundaran. 2001. Purification of sandal spike Seemüller, E., C. Marcone, U. Lauer, A. Ragozzino and M. Göschl. 1998. phytoplasma for the production of polyclonal antibody. Curr. Sci. 80: Current status of molecuar classification of the phytoplasmas. J. Plant 1489–1494. Pathol. 80: 3–26. Toth, K.F., N. Harrison and B.B. Sears. 1994. Phylogenetic relation- Seemüller, E., M. Garnier and B. Schneider. 2002. Mycoplasmas of ships among members of the class Mollicutes deduced from rps3 gene plants and insects. In Molecular Biology and Pathogenicity of Myco- sequences. Int. J. Syst. Bacteriol. 44: 119–124. plasmas (edited by Razin and Hermann). Kluwer Academic/Plenum Tran-Nguyen, L.T. and K.S. Gibb. 2006. Extrachromosomal DNA iso- Publishers, Dordrecht, The Netherlands, pp. 91–116. lated from tomato big bud and Candidatus Phytoplasma australiense Seemüller, E. and B. Schneider. 2004. ‘Candidatus Phytoplasma mali’, phytoplasma strains. Plasmid 56: 153–166. ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma pruno- Tran-Nguyen, L.T., M. Kube, B. Schneider, R. Reinhardt and K.S. Gibb. rum’, the causal agents of apple proliferation, pear decline and 2008. Comparative genome analysis of “Candidatus Phytoplasma aus- European stone fruit yellows, respectively. Int. J. Syst. Evol. Microbiol. traliense” (subgroup tuf-Australia I; rp-A) and “Ca. Phytoplasma ast- 54: 1217–1226. eris” strains OY-M and AY-WB. J. Bacteriol. 190: 3979–3991. Shen, W.C. and C.P. Lin. 1993. Production of monoclonal antibod- Tsai, J.H. 1979. Vector transmission of mycoplasmal agents of plant dis- ies against a mycoplasmalike organism associated with sweetpotato eases. In The Mycoplasmas, vol. III, Plant and Insect Mycoplasmas witches’ broom. Phytopathology 83: 671–675. (edited by Whitcomb and Tully). Academic Press, New York, pp. Shen, W.C. and C.P. Lin. 1994. Application of immunofluorescent staining, 266–307. tissue blotting techniques against a mycoplasmalike organism assoicated Valiunas, D., J. Staniulis and R.E. Davis. 2006. ‘Candidatus Phyto- with sweetpotato witches’ broom. Plant Pathol. Bull. 3: 79–83. plasma fragariae’, a novel phytoplasma taxon discovered in yellows Siddique, A.B.M., J.N. Giuthrie, K.B. Walsh, D.T. White and P.T. Scott. diseased strawberry, Fragaria x ananassa. Int. J. Syst. Evol. Microbiol. 1998. Histopathology and within-plant distribution of the phyto- 56: 277–281. plasma associated with Australian papaya dieback. Plant Dis. 82: Van Helden, M., W.F. Tjallinghii and T.A. Van Beek. 1994. Phloem col- 1112–1120. lection from lettuce (Lactuca sativa L.): Chemical comparison among Siller, W., B. Kuhbandner, R. Marwitz, H. Petzold and E. Seemüller. collection methods. J. Chem. Ecol. 20: 3191–3206. 1987. Occurrence of mycoplasma-like organisms in parenchyma cells Verdin, E., P. Salar, J.L. Danet, E. Choueiri, F. Jreijiri, S. El Zammar, of Cuscuta odorata (Ruiz et Pav.). J. Phytopathol. 119: 147–159. B. Gelie, J.M. Bové and M. Garnier. 2003. ‘Candidatus Phytoplasma Sinclair, W.A., H.M. Griffiths and I.M. Lee. 1994. Mycoplasmalike phoenicium’ sp. nov., a novel phytoplasma associated with an emerg- organisms as causes of slow growth and decline of trees and shrubs. ing lethal disease of almond trees in Lebanon and Iran. Int. J. Syst. J. Arboric. 20: 176–189. Evol. Microbiol. 53: 833–838. Sinha, R.C. and E.A. Peterson. 1972. Uptake and persistence of oxytet- Wagner, M., C. Fingerhut, H.J. Gross and A. Schön. 2001. The racycline in aster plants and vector leafhoppers in relation to inhibi- first phytoplasma RNase P RNA provides new insights into the tion of clover phyllody agent. Phytopathology 62: 377–383. sequence requirements of this ribozyme. Nucleic Acids Res. 29: Sinha, R.C. 1979. Purification and serology of mycoplasma-like organ- 2661–2665. isms from aster yellows-infected plants. Can. J. Plant Pathol. 1: 65–70. Wang, K. and C. Hiruki. 2000. Heteroduplex mobility assay detects DNA Sjölund, R.D. 1997. The phloem sieve element: a river runs through it. mutations for differentiation of closely related phytoplasma strains. Plant Cell 9: 1137–1146. J. Microbiol. Methods 41: 59–68. Smart, C.D., B. Schneider, C.L. Blomquist, L.J. Guerra, N.A. Harrison, Waters, H. and P. Hunt. 1980. The in vivo three-dimensional form of U. Ahrens, K.H. Lorenz, E. Seemuller and B.C. Kirkpatrick. 1996. a plant mycoplasma-like organism revealed by the analysis of serial Phytoplasma-specific PCR primers based on sequences of the 16S– ultra-thin sections. J. Gen. Microbiol. 116: 111–131. 23S rRNA spacer region. Appl. Environ. Microbiol. 62: 2988–2993. Webb, D.R., R.G. Bonfiglioli, L. Carraro, R. Osler and R.H. Symons. Smith, A.J., R.E. McCoy and J.H. Tsai. 1981. Maintenance in vitro of 1999. Oligonucleotides as hybridization probes to localize phy- the aster yellows mycoplasmalike organism. Phytopathology 71: toplasmas in host plants and insect vectors. Phytopathology 89: 819–822. 894–901. Order IV. Anaeroplasmatales 719

Wei, W., S. Kakizawa, H.Y. Jung, S. Suzuki, M. Tanaka, H. Nishigawa, S. Whitcomb, R.F., D.D. Jensen and J. Richardson. 1966a. The infection of Miyata, K. Oshima, M. Ugaki, T. Hibi and S. Namba. 2004a. An anti- leafhoppers by western X-disease. virus: II. Fluctuation of virus con- body against the SecA membrane protein of one phytoplasma reacts centration in the hemolymph after injection. Virology 28: 454–458. with those of phylogenetically different phytoplasmas. Phytopatho­ Whitcomb, R.F., D.D. Jensen and J. Richardson. 1966b. The infection of logy 94: 683–686. leafhoppers by the western X-disease virus: I. Frequency of transmis- Wei, W., S. Kakizawa, S. Suzuki, H.Y. Jung, H. Nishigawa, S. Miyata, sion after injection or acquisition feeding. Virology 28: 448–453. K. Oshima, M. Ugaki, T. Hibi and S. Namba. 2004b. In planta dynamic White, D.T., L.L. Blackall, P.T. Scott and K.B. Walsh. 1998. Phylogenetic analysis of onion yellows phytoplasma using localized inoculation by positions of phytoplasmas associated with dieback, yellow crinkle and insect transmission. Phytopathology 94: 244–250. mosaic diseases of papaya, and their proposed inclusion in ‘Candida- Wei, W., R.E. Davis, I.M. Lee and Y. Zhao. 2007. Computer-simulated tus Phytoplasma australiense’ and a new taxon, ‘Candidatus Phyto- RFLP analysis of 16S rRNA genes: identification of ten new phyto- plasma australasia’. Int. J. Syst. Bacteriol. 48: 941–951. plasma groups. Int. J. Syst. Evol. Microbiol. 57: 1855–1867. Wongkaew, P. and J. Fletcher. 2004. Sugarcane white leaf phytoplasma Wei, W., R.E. Davis, R. Jomantiene and Y. Zhao. 2008a. Ancient, recur- in tissue culture: long-term maintenance, transmission, and oxytetra- rent phage attacks and recombination shaped dynamic sequence- cycline remission. Plant Cell Rep. 23: 426–434. variable mosaics at the root of phytoplasma genome evolution. Proc. Yu, Y.L., K.W. Yeh and C.P. Lin. 1998. An antigenic protein gene of a Natl. Acad. Sci. U. S. A. 105: 11827–11832. phytoplasma associated with sweet potato witches’ broom. Microbiol- Wei, W., I.M. Lee, R.E. Davis, X. Suo and Y. Zhao. 2008b. Automated ogy 144: 1257–1262. RFLP pattern comparison and similarity coefficient calculation for Zhao, Y., Q. Sun, W. Wei, R.E. Davis, W. Wu and Q. Liu. 2009. ‘Candi- rapid delineation of new and distinct phytoplasma 16Sr subgroup datus Phytoplasma tamaricis’, a novel taxon discovered in witches’- lineages. Int. J. Syst. Evol. Microbiol. 58: 2368–2377. broom-diseased salt cedar (Tamarix chinensis Lour.). Int. J. Syst. Evol. Weintraub, P.G. and L. Beanland. 2006. Insect vectors of phytoplasmas. Microbiol. 59: 2496–2504. Annu. Rev. Entomol. 51: 91–111. Zreik, L., P. Carle, J.M. Bové and M. Garnier. 1995. Characterization Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Man- of the mycoplasmalike organism associated with witches’ broom dis- delco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic ease of lime and proposition of a Candidatus taxon for the organ- analysis of the mycoplasmas: basis for their classification. J. Bacteriol. ism, “Candidatus Phytoplasma aurantifolia”. Int. J. Syst. Bacteriol. 45: 171: 6455–6467. 449–453.

Order IV. Anaeroplasmatales Robinson and Freundt 1987, 81VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.na.e.ro.plas.ma.ta¢les. N.L. neut. n. Anaeroplasma, -atos type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Anaeroplasmatales the Anaeroplasma order.

This order in the class Mollicutes represents a unique group of anaeroplasmas exist in a natural environment where the oxi- strictly anaerobic, wall-less prokaryotes (trivial name, anaero- dation potential is maintained at a low level by the metabo- plasmas) first isolated from the bovine and ovine rumen. lism of associated micro-organisms. Anaerobic methods for Other than their anaerobiosis, the description of organisms preparing media and culture techniques for the organisms in the order is essentially the same as for the class. A single are essentially those described by Hungate (1969), with family, Anaeroplasmataceae, with two genera, was proposed media and inocula maintained in closed vessels and expo- to recognize the two most prominent characteristics of the sure to air avoided during inoculation and incubation. A organisms: a requirement of sterol supplements for growth by primary isolation medium and clarified rumen fluid broth those strictly anaerobic organisms now assigned to the genus have been described (Bryant and Robinson, 1961; Robinson, Anaeroplasma; and strictly anaerobic growth in the absence of 1983; Robinson et al., 1975). sterol supplements by those now assigned to the genus Aster- oleplasma. Genome sizes range from 1542 to 1794 kbp as esti- References mated by renaturation kinetics. The DNA G+C content ranges Bryant, M.P. and I.M. Robinson. 1961. An improved nonselective cul- from 29 to 40 mol%. All species examined utilize the univer- ture medium for ruminal bacteria and its use in determining diur- sal genetic code in which UGA is a stop codon. Phylogenetic nal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44: studies indicate that members of the Anaeroplasmatales are 1446–1456. Hungate, R.E. 1969. A roll tube method for cultivation of strict anaer- much more closely related to the Acholeplasmatales than to the obes. In Methods in Microbiology, vol. 3B (edited by Norris and Mycoplasmatales or Entomoplasmatales (Weisburg et al., 1989). ­Ribbons). Academic Press, London, pp. 117–132. Type genus: Anaeroplasma Robinson, Allison and Hartman Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abac- AL 1975, 179 . toclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from rumen. Int. J. Syst. Bacteriol. 25: 173–181. Further descriptive information Robinson, I.M. 1983. Culture media for anaeroplasmas. In Methods in The initial proposal for elevation of the anaeroplasmas to an Mycoplasmology, vol. 1, (edited by Razin and Tully). Academic Press, order of the class Mollicutes (Robinson and Freundt, 1987) New York, pp. 159–162. was based upon the description of three novel species and the Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended observation that some anaeroplasmas did not have a sterol classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81. requirement for growth. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Mandelco, The obligate requirement for anaerobic growth condi- J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analy- tions is the single most important property in distinguishing sis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: members of the Anaeroplasmatales from other mollicutes. The 6455–6467. 720 Family I. Anaeroplasmataceae

Family I. Anaeroplasmataceae Robinson and Freundt 1987, 80VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.na.e.ro.plas.ma.ta.ce¢ae. N.L. neut. n. Anaeroplasma, -atos type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Anaeroplasmataceae the Anaeroplasma family. All members have an obligate requirement for anaerobiosis. Further descriptive information Organisms assigned to the genus Anaeroplasma require sterol The obligate requirement for anaerobic growth conditions and supplements for growth. Organisms assigned to the genus Aster- for growth only in media containing cholesterol is established oleplasma grow in the absence of sterol supplements. Other with the methods described by Hungate (1969), with media characteristics are as described for the type genus. and inocula maintained in closed vessels and exposure to air Type genus: Anaeroplasma Robinson, Allison and Hartman avoided during inoculation and incubation. The primary isola- 1975, 179AL. tion medium and the clarified rumen fluid broth supplemented with cholesterol have been described (Robinson, 1983).

Genus I. Anaeroplasma Robinson, Allison and Hartman 1975, 179AL

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k Jo h a n s s o n A.na.e.ro.plas¢ma. Gr. prefix an without; Gr. masc. n. aer air; Gr. neut. n. plasma a form; N.L. neut. n. Anaero- plasma intended to denote “anaerobic mycoplasma”. Cells are predominantly coccoid, about 500 nm in diameter; Robinson and Hungate, 1973). Anaerobic mollicutes in a sew- clusters of up to ten coccoid cells may be joined by short fila- age sludge digester were cultured in an anaerobic cabinet ments. Older cells have a variety of pleomorphic forms. Cells (Rose and Pirt, 1981). Although it is possible that other types lack a cell wall and are bound by a single plasma membrane. of anaerobic culture techniques might be acceptable (anaero- Gram-stain-negative due to absence of cell wall. Obligately bic culture jar or GasPak system), the effective use of such anaerobic; the inhibitory effect of oxygen on growth is not equipment has not been demonstrated (Robinson, 1983). alleviated during repeated subcultures. Require sterol supple- Strains with bacteriolytic activity are detected with the addi- ments for growth. Nonmotile. Optimal temperature, 37°C; no tion of autoclaved Escherichia coli cells to the Primary Isolation growth at 26 or 47°C. Optimal pH, 6.5–7.0. Surface colonies Medium (PIM) described below. Clear zones around colonies have a dense center with a translucent periphery, or “fried-egg” of anaeroplasmas, when viewed by a stereoscopic microscope, appearance. Subsurface colonies are golden, irregular, and are suggestive of bacteriolytic anaeroplasmas. Colonies can be often multilobed. Strains vary in their ability to ferment vari- subcultured to clarified rumen fluid broth (CRFB) medium ous carbohydrates. The products of carbohydrate fermentation described below. include acids (generally acetic, formic, propionic, lactic, and A slide agglutination test was first used to show that the anti- succinic), ethanol, and gases (primarily CO2, but some strains gens of anaerobic mollicutes were not related to established also produce H2). Bacteriolytic and nonbacteriolytic strains have Mycoplasma or Acholeplasma species found in cattle (Robinson been described. Commensals in the bovine and ovine rumen. and Hungate, 1973). Later, the agglutination test was adapted

DNA G+C content (mol%): 29–34 (Tm, Bd). to either a plate or tube test and combined with an agar gel Type species: Anaeroplasma abactoclasticum Robinson, Allison diffusion test and a modified growth inhibition procedure to and Hartman 1975, 179AL. examine the antigenic interrelationships among the anaero- bic mollicutes (Robinson and Rhoades, 1977). On the basis Further descriptive information of these tests, a serological grouping of anaerobic mollicutes Cells of Anaeroplasma examined by phase-contrast microscopy appeared compatible with the group separations based upon appear as single cells, clumps, dumbbell forms, and clusters cultural, biochemical, and biophysical properties of the organ- of coccoid forms joined by short filaments. In electron micro- isms (Robinson, 1979; Robinson and Rhoades, 1977). graphs of negatively stained preparations, pleomorphic forms There is no current evidence for the pathogenicity of any of are observed; these include filamentous cells, budding cells, the Anaeroplasma species described so far. Obligately anaerobic and cells with bleb-like structures. mollicutes appear to be a heterogeneous group that has been All species examined have similar fermentation products of found so far only in the rumen of cattle and sheep (Robinson, acetate, formate, lactate, ethanol, and carbon dioxide (Robin- 1979; Robinson et al., 1975). Each new isolated group of these son et al., 1975). Anaeroplasma abactoclasticum is the only species organisms seems to have different properties, suggesting that known not to digest casein. Anaeroplasma abactoclasticum strains additional undescribed species are likely to exist. The ecological are the only ones known to produce succinate through fermen- role of these organisms in the rumen has not been determined. tation. Anaeroplasma bactoclasticum, Anaeroplasma intermedium, Although the titer of these organisms in the rumen appears and Anaeroplasma varium are the only species known to produce to be low when compared to titers of other rumen organisms, hydrogen and propionate during their fermentation. the mollicutes probably contribute to the pool of microbial The roll-tube anaerobic culture technique (Hungate, fermentation products at that site. Growth of anaeroplasmas is 1969), with pre-reduced medium maintained in a system for inhibited by thallium acetate (0.2%), bacitracin (1000 mg/ml), exclusion of oxygen, is used to culture the organisms (Robin- streptomycin (200 mg/ml), and d-cycloserine (500 mg/ml), son, 1983; Robinson and Allison, 1975; Robinson et al., 1975; but not by benzylpenicillic acid (1000 U/ml). Genus I. Anaeroplasma 721

Enrichment and isolation procedures thought to lack a sterol requirement for growth. Later, when other obligately anaerobic mollicutes were isolated, these and The PIM medium used to grow and detect anaerobic mycoplas- the JRT strain were found to require sterol for growth. A pro- mas (Robinson, 1983; Robinson et al., 1975; Robinson and Hun- posal was then made to form the new genus Anaeroplasma to gate, 1973) contains: 40% (v/v) rumen fluid strained through accommodate strain JRT (as Anaeroplasma bactoclasticum; Rob- cheesecloth, autoclaved, and clarified by centrifugation; 0.05% inson and Allison, 1975) and a second anaerobic mollicute (w/v) glucose; 0.05% (w/v) cellobiose; 0.05% (w/v) starch; ­designated Anaeroplasma abactoclasticum (Robinson et al., 1975). 3.75% (v/v) of a mineral solution consisting of 1.7 × 10−3 M These developments prompted a proposal for an amended clas- K HPO , 1.3 × 10−3 M KH PO , 7.6 × 10−4 M NaCl, 3.4 × 10−3 M 2 4 2 4 sification of anaerobic mollicutes, which included descriptions (NH ) SO , 4.1 × 10−4 M CaCl , and 3.8 × 10−4 M MgSO ·7H O; 4 2 4 2 4 2 of Anaeroplasma varium and Anaeroplasma intermedium, the family 0.2% (w/v) trypticase; 0.1% (w/v) yeast extract; 0.0001% (w/v) Anaeroplasmataceae, and the order Anaeroplasmatales within the resazurin; 0.5% (w/v) autoclaved Escherichia coli cells; 0.4% (w/v) class Mollicutes (Robinson and Freundt, 1987). Na CO ; 0.05% (w/v) cysteine hydrochloride; 1.5% (w/v) agar; 2 3 Early serological studies suggested the existence of several and 0.0006% (w/v) benzylpenicillic acid. Pure cultures are estab- distinct species of anaeroplasmas. Subsequent reports on DNA– lished by picking individual colonies from PIM roll tubes and sub- DNA hybridization, DNA base composition, and genome size culturing into CRFB medium. CRFB medium contains the same comparisons of organisms in the group also indicated the exis- ingredients and concentrations as PIM, except: glucose, cello- tence of a number of species in two distinct genera of anaero- biose, and starch concentrations are 0.2% (w/v), and autoclaved bic mollicutes (Christiansen et al., 1986; Stephens et al., 1985). Escherichia coli cells, agar, and benzylpenicillic acid are omitted. Strains initially assigned to Acholeplasma abactoclasticum [serovar Growth also occurs in a rumen fluid-free medium (Medium D) 3, type strain 6-1T (=ATCC 27879T)] were found to be a single spe- in which growth factors supplied in rumen fluid are replaced by cies with about 80% interstrain DNA–DNA hybridization. How- lipopolysaccharide (Boivin; Difco) and cholesterol (Robinson, ever, strains A-2T (serovar 1) and 7LAT (serovar 2), previously 1983; Robinson et al., 1975), or in a completely defined medium included in the description of Acholeplasma bactoclasticum, are the in which the trypticase, yeast extract, and lipopolysaccharide of type strains of separate species designated Anaeroplasma varium Medium D are replaced by amino acids, vitamins, and phosphati- and Anaeroplasma intermedium, respectively (Robinson and Fre- dylcholine esterified with unsaturated fatty acids (Robinson, undt, 1987). Strains of Anaeroplasma all have DNA G+C contents 1979, 1983). An agar-overlay plating technique carried out in an in the range 29.3–33.7 mol%, whereas the base composition of anaerobic hood has also been reported to be an effective isola- serovar 4 strains 161T, 162, and 163 clustered above 40 mol%. tion procedure (Robinson, 1979). Anaerobic mollicutes grow These were assigned to the new genus Asteroleplasma [type strain only in a prereduced medium maintained in a system for exclu- 161T (=ATCC 27880T)] whose members are ­anaerobic, but do sion of oxygen. When resazurin is used in the test medium and not require sterol for growth. Genome sizes ranged from 1542 becomes oxidized, mollicutes will fail to grow. to 1715 kbp for Anaeroplasma species, as determined by renatur- Maintenance procedures ation kinetics (Christiansen et al., 1986). Although the genome Cultures are viable after storage for as long as 5 years at −40°C in sizes reported were in the expected range for members of the CRFB medium. They may also be preserved by lyophilization using class Mollicutes, no data are currently available on genome sizes standard techniques for other mollicutes (Leach, 1983). However, estimated by the more accurate pulsed-field gel electrophoresis the type strains of several species of Anaeroplasma are no longer technique. A phylogenetic analysis of members of the Anaero- available from the American Type Culture Collection because it plasmatales, based upon 16S rRNA gene sequence comparison, was impossible to revive the cultures sent by the depositors. was carried out by Weisburg et al. (1989). Anaeroplasma and Acholeplasma are sister genera basal on the mollicute tree. Differentiation of the genus Anaeroplasma Acknowledgements from other genera The major contributions to the foundation of this material by Properties that partially fulfill criteria for assignment to the Joseph G. Tully are gratefully acknowledged. class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene Further reading sequences. The obligately anaerobic nature of Anaeroplasma spe- Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular cies is a distinctive and stable characteristic among these organ- Biology and Pathogenicity of Mycoplasmas (edited by Razin isms. Strictly anaerobic growth plus the requirement for sterol and Herrmann). Kluwer Academic/Plenum Publishers, New supplements for growth exclude assignment to any other taxon York, pp. 1–29. in the class. Moreover, the bacteriolytic capability possessed by some of the anaeroplasmas has not been reported for other Differentiation of the species of the genus Anaeroplasma mycoplasmas. Plasmalogens (alkenyl-glycerol ethers), which are found in various anaerobic bacteria but not in aerobic bac- The technical challenges of cultivating these anaerobic molli- teria, are major components of polar lipids from anaeroplasmas cutes have led to a current reliance principally on the combina- (Langworthy et al., 1975); this further supports the contention tion of 16S rRNA gene sequencing and reciprocal serology for that anaeroplasmas are distinct from other mollicutes. species differentiation. Serological characterization of anaero- plasmas has been performed with agglutination, ­modified Taxonomic comments metabolism inhibition, and immunodiffusion tests (Robinson The first organism in the group to be described was referred to and Rhoades, 1977). Failure to cross-react with antisera against as Acholeplasma bactoclasticum (type strain JRT = ATCC 27112T; previously recognized species provides substantial evidence for Robinson and Hungate, 1973) because the organism was species novelty. DNA–DNA hybridization values between species 722 Family I. Anaeroplasmataceae examined are less than 5%. Bacteriolytic and nonbacteriolytic due to lysis of the suspended cells by a diffusible enzyme(s). organisms occur within the genus. When grown on agar media On media lacking suspended cells, bacteriolytic and nonbac- containing a suspension of autoclaved Escherichia coli cells, teriolytic strains of Anaeroplasma cannot be distinguished from bacteriolytic strains form colonies surrounded by a clear zone each other on the basis of colonial or cellular morphology.

List of species of the genus Anaeroplasma 1. Anaeroplasma abactoclasticum Robinson, Allison and Hart- that attacks the peptidoglycan layer of the cell wall. Shares man 1975, 179AL some serological relationship to other established species in a.bac.to.clas¢ti.cum. Gr. pref. a without; Gr. bakt- (L. trans- the genus, but can be distinguished by agglutination, modi- literation bact-) part of the stem of the Gr. dim. n. bakterion fied metabolism inhibition, and agar gel immunodiffusion (L. transliteration bacterium) a small rod; N.L. adj. clasticus, precipitation tests. -a, um (from Gr. adj. klastos, -ê, -on broken in pieces) break- No evidence of pathogenicity. ing; N.L. neut. adj. abactoclasticum intended to denote “not Source: occurs in the bovine and ovine rumen. bacteriolytic”. DNA G+C content (mol%): 32.5 to 33.7 (Tm, Bd). Type strain: JR, ATCC 27112. This is the type species of the genus Anaeroplasma. Cells Sequence accession no. (16S rRNA gene): M25049. are coccoid, about 500 nm in diameter, sometimes joined into short chains by filaments. Colonies on solid medium 3. Anaeroplasma intermedium Robinson and Freundt 1987, 79VP are subsurface, but nevertheless present a typical fried-egg in.ter.me¢di.um. L. neut. adj. intermedium intermediate. appearance. Growth is inhibited by 20 mg/ml digitonin. A major distinguishing factor is the lack of the extracellular Cellular morphology and colonial features are similar to bacterioclastic and proteolytic enzymes that characterize the those of Acholeplasma bactoclasticum. Serologically distinct lytic species. from other species in the genus by agglutination, metabo- No evidence of a role in pathogenicity. lism inhibition, and agar gel immunodiffusion precipitation Source: occurs primarily in the bovine and ovine rumen. tests (Robinson and Rhoades, 1977). DNA G+C content (mol%): 29.3 (Bd). No evidence of pathogenicity. Type strain: 6-1, ATCC 27879. Source: occurs in the bovine and ovine rumen. Sequence accession no. (16S rRNA gene): M25050. DNA G+C content (mol%): 32.5 (Bd). Type strain: 7LA, ATCC 43166. 2. Anaeroplasma bactoclasticum (Robinson and Hungate Sequence accession no. (16S rRNA gene): not available. 1973) Robinson and Allison 1975, 186AL (Acholeplasma bacto- VP clasticum Robinson and Hungate 1973, 180) 4. Anaeroplasma varium Robinson and Freundt 1987, 79 ¢ bac.to.clas¢ti.cum. Gr. bakt- (L. transliteration bact-) part of va ri.um. L. neut. adj. varium diverse, varied, intended to the stem of the Gr. dim. n. bakterion (L. transliteration bac- mean different from Anaeroplasma bactoclasticum. terium) a small rod; N.L. adj. clasticus, -a, um (from Gr. adj. Cellular morphology and colonial features are similar to klastos, -ê, -on broken in pieces) breaking; N.L. neut. adj. bac- those of Acholeplasma bactoclasticum. Serologically distinct toclasticum bacteria-breaking. from other species in the genus by agglutination, metabo- Pleomorphic and coccoid cells ranging in size from 550 lism inhibition, and agar gel immunodiffusion precipitation to 2000 nm in diameter. Cells cluster and sometimes form tests (Robinson and Rhoades, 1977). short chains. Colonies on solid medium have a typical fried- No evidence of pathogenicity. egg appearance. Optimal temperature for growth is between Source: occurs in the bovine and ovine rumen. 30 and 47°C. Growth is inhibited by 20 mg/ml digitonin. DNA G+C content (mol%): 33.4 (Tm). Skim milk is cleared by a proteolytic, extracellular enzyme Type strain: A-2, ATCC 43167. and certain bacteria are lysed by an extracellular enzyme Sequence accession no. (16S rRNA gene): M23934.

Genus II. Asteroleplasma Robinson and Freundt 1987, 79VP

Da n i e l R. Br o w n , Ja n e t M. Br a db u r y a n d Ka r l -Er i k j o h a n s s o n A.ste.rol.e.plas¢ma. Gr. pref. a not; N.L. neut. n. sterolum sterol; e combining vowel; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Asteroleplasma name intended to indicate that sterol is not required for growth. Cellular and colonial morphology similar to species of the Type species: Asteroleplasma anaerobium Robinson and Fre- genus Anaeroplasma. Nonmotile. The three strains that form undt 1987, 79VP. the new genus and species are obligately anaerobic and capable of growth in the absence of cholesterol or serum supplements. Further descriptive information Temperature optimum for growth is 37°C. No evidence of bac- The most prominent characteristics of the organisms are strictly teriolytic activity. The organisms are serologically distinct from anaerobic growth and growth in the absence of sterol supple- other members in the family Anaeroplasmataceae. Occur in the ments. The G+C contents of strains analyzed to date are higher ovine rumen. than the values for Anaeroplasma species (Stephens et al., 1985). T DNA G+C content (mol%): about 40 (Tm, Bd). DNA–DNA reassociation values clearly show that strains 161 , Genus II. Asteroleplasma 723

162, and 163 of Asteroleplasma anaerobium are genetically related et al., 1975), were serologically distinct (Robinson and Rhoades, and distinct from established species in the genera Acholeplasma 1977), and had G+C contents that were much higher than those or Anaeroplasma (Stephens et al., 1985). Tube agglutination of Anaeroplasma species (Christiansen et al., 1986). Less than 5% tests and gel diffusion precipitation tests showed that strains DNA–DNA relatedness existed between these strains and spe- assigned to Asteroleplasma anaerobium are serologically distinct cies assigned to the genus Anaeroplasma (Stephens et al., 1985). from Anaeroplasma species (Robinson and Rhoades, 1977). No Lastly, the significance of the group and the need to clarify its data have been reported on antibiotic sensitivity or pathoge- taxonomic status was emphasized when it was demonstrated nicity of asteroleplasmas. Strains have been isolated only from that a significant proportion of the anaerobic mollicute popula- sheep rumen. Isolation and maintenance techniques are simi- tion in the bovine and ovine rumen does not require sterol for lar to those reported for Anaeroplasma species. growth (Robinson and Rhoades, 1982). The phylogenetic analy- sis of Weisburg et al. (1989) indicated that Asteroleplasma anaero- Differentiation of the genus Asteroleplasma bium had branched from the Firmicutes lineage independently from other genera of Acholeplasma and Anaeroplasma. Further, Asteroleplasma shared Properties that partially fulfill criteria for assignment to the two of three important synapomorphies that united the Myco- class Mollicutes (Brown et al., 2007) include absence of a cell plasma and Spiroplasma lineages. Thus, the question of possible wall, filterability, and the presence of conserved 16S rRNA gene monophyly and the true phylogenetic position of Asteroleplasma sequences. The obligately anaerobic nature of Asteroleplasma is with respect to other mollicutes remains open. a distinctive and stable characteristic. Strictly anaerobic growth Acknowledgements plus growth in the absence of sterol supplements exclude assign- ment to any other taxon in the class. Extracellular bacteriolytic The major contributions to the foundation of this material by and proteolytic enzymes are absent. Growth is not inhibited by Joseph G. Tully are gratefully acknowledged. 20 mg/ml digitonin. Further reading Taxonomic comments Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular The taxonomic position of strains 161T, 162, and 163 of obligately Biology and Pathogenicity of Mycoplasmas (edited by Razin anaerobic mollicute serovar 4 was delineated through obser- and Herrmann). Kluwer Academic/Plenum Publishers, New vations that they did not require sterol for growth (Robinson York, pp. 1–29.

List of species of the genus Asteroleplasma 1. Asteroleplasma anaerobium Robinson and Freundt 1987, 79VP serological agglutination, metabolism inhibition, and agar a.na.e.ro¢bi.um. Gr. pref. an not; Gr. n. aer air; Gr. n. bios life; gel immunodiffusion precipitation tests. N.L. neut. adj. anaerobium not living in air. No evidence of pathogenicity. Source: all isolates have been identified from the bovine or This is the type species of the genus Asteroleplasma. Cell ovine rumen. morphology and colonial characteristics are similar to those DNA G+C content (mol%): 40.2–40.5 (Bd, T ). of other members of the order Anaeroplasmatales. Strains 161T, m Type strain: 161 (the type strain ATCC 27880 no longer 162, and 163 form a homogeneous and distinct serological exists). group, as judged by about 80% DNA–DNA ­hybridization and Sequence accession no. (16S rRNA gene): M22351.

References Robinson, I.M. and K.R. Rhoades. 1977. Serological relationships between strains of anaerobic mycoplasmas. Int. J. Syst. Bacteriol. 27: 200–203. Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal stan- Robinson, I.M. 1979. Special features of anaeroplasmas. In The Myco- dards for description of new species of the class Mollicutes (division plasmas, vol. 1 (edited by Barile and Razin). Academic Press, New Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719. York, pp. 515–528. Christiansen, C., E.A. Freundt and I.M. Robinson. 1986. Genome size Robinson, I.M. and K.R. Rhoades. 1982. Serologic relationships between and deoxyribonucleic acid base composition of Anaeroplasma abac- strains of anaerobic mycoplasmas. Rev. Infect. Dis. 4: S271. toclasticum, Anaeroplasma bactoclasticum, and a sterol-nonrequiring Robinson, I.M. 1983. Culture media for anaeroplasmas. In Methods in anaerobic mollicute. Int. J. Syst. Bacteriol. 36: 483–485. Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, Hungate, R.E. 1969. A roll tube method for cultivation of strict anaer- New York, pp. 159–162. obes. In Methods in Microbiology, vol. 3B (edited by Norris and Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended ­Ribbons). Academic Press, London, pp. 117–132. classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: Langworthy, T., W. Mayberry, P. Smith and I. Robinson. 1975. Plasmalo- 78–81. gen composition of Anaeroplasma. J. Bacteriol. 122: 785–787. Robinson, J.P. and R.E. Hungate. 1973. Acholeplasma bactoclasticum sp. Leach, R.H. 1983. Preservation of Mycoplasma cultures and culture col- n., an anaerobic mycoplasma from the bovine rumen. Int. J. Syst. lections. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Bacteriol. 23: 171–181. Tully). Academic Press, New York, pp. 197–204. Rose, C. and S. Pirt. 1981. Conversion of glucose to fatty acids and meth- Robinson, I.M. and M.J. Allison. 1975. Transfer of Acholeplasma bactoclas- ane: roles of two mycoplasmal agents. J. Bacteriol. 147: 248–254. ticum Robinson and Hungate to genus Anaeroplasma (Anaeroplasma Stephens, E., I. Robinson and M. Barile. 1985. Nucleic acid relationships bactoclasticum Robinson and Hungate comb. nov.), emended descrip- among the anaerobic mycoplasmas. J. Gen. Microbiol. 131: 1223–1227. tion of species. Int. J. Syst. Bacteriol. 25: 182–186. Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. ­Mandelco, Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abac- J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analy- toclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from sis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: rumen. Int. J. Syst. Bacteriol. 25: 173–181. 6455–6467.